molecules

Article Solid-Phase Synthesis of Phosphorothioate/ Phosphonothioate and / Phosphonamidate Oligonucleotides

OndˇrejKostov *, Radek Liboska, OndˇrejPáv *, Pavel Novák and Ivan Rosenberg *

Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; [email protected] (R.L.); [email protected] (P.N.) * Correspondence: [email protected] (O.K.); [email protected] (O.P.); [email protected] (I.R.); Tel.: +420-220-183-381 (I.R.)  Received: 1 May 2019; Accepted: 14 May 2019; Published: 15 May 2019 

Abstract: We have developed a robust solid-phase protocol which allowed the synthesis of chimeric oligonucleotides modified with phosphodiester and O-methylphosphonate linkages as well as their P-S and P-N variants. The novel O-methylphosphonate-derived modifications were obtained by oxidation, sulfurization, and amidation of the O-methyl-(H)- internucleotide linkage introduced into the oligonucleotide chain by H- chemistry using nucleoside-O-methyl-(H)- as monomers. The H-phosphonate coupling followed by oxidation after each cycle enabled us to successfully combine H-phosphonate and chemistries to synthesize diversely modified oligonucleotide strands.

Keywords: H-phosphinate; H-phosphonate; phosphoramidite; oligonucleotide; phosphonate; phosphorothioate; phosphonothioate; phosphoramidate; phosphonamidate

1. Introduction

Recently,we published a detailed study on the influence of the incorporation of 20-deoxy-nucleoside 30-O- and 50-O-methylphosphonate units on the hybridization properties of modified DNA strands and on their ability to elicit E. coli RNase H activity in heteroduplexes [1]. Although the insertion of the bridging –CH2– group into the phosphodiester linkage should increase the total entropy of the system due to an additional degree of freedom, leading to duplex destabilization, only the incorporation of 30-O-methylphosphonate units into the DNA strand decreased the stability of the appropriate heteroduplexes. In contrast, the presence of 50-O-methylphosphonate units slightly stabilized the modifDNA*RNA heteroduplexes. These oligonucleotides, known as MethylPhosphonate Nucleic Acids (MePNA), when they contained various ratios of nucleoside-50- and 50-O-methyl-phosphonate units in an alternating mode exhibited superior enhancement of the RNase H cleavage rate. To improve the synthesis of MePNA, we developed the straightforward synthesis of nucleoside-O-methyl-(H)-phosphinates and 50-deoxynucleoside-50-S-methyl-(H)-phosphinates [2,3] that were closely related to the well-known nucleoside H-, [4,5] and demonstrated their compatibility with the H-phosphonate chemistry of the oligonucleotide synthesis. Recently, Herdewijn [6] has exploited this methodology to introduce various nucleoside phosphonates into oligonucleotides. In this study, we present the extension of our methodology on the synthesis of modified oligonucleotides by a combination of phosphoramidite and H-phosphonate chemistries using nucleoside phosphoramidite, and H-phosphonate 1, nucleoside-O-methyl-(H)-phosphinate, and

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Molecules 2019, 24, x FOR PEER REVIEW 2 of 11

50-deoxynucleoside-50-S-methyl-(H)-phosphinate monomers 2 as building blocks (Figure1), respectively.respectively. Standard Standard p phosphoramiditehosphoramidite chemistry chemistry allows allows the the synthesis synthesis of phosphatephosphate 3,, and and phosphorothioatephosphorothioate4 internucleotide4 internucleotide linkages. linkageH-phosphonates. H-phosphonate chemistry chemistry affords evenaffords wider even possibilities wider sincepossibilities the H-phosphonate since the H- internucleotidephosphonate internucleotide linkage in 5 can linkage be oxidized, in 5 can sulfurized, be oxidized, or amidated sulfurized, to form or appropriateamidated to phosphateform appropriate3 [7,8], phosphate phosphorothioate 3 [7,8], phosphorothioate4 [9] or phosphoramidate 4 [9] or phosphor6 [10] internucleotideamidate 6 [10] linkagesinternucleotide (Figure linkages1). To be(Figure able to1). synthesize To be able oligonucleotides to synthesize modified oligonucleotides with phosphodiester, modified with phosphodiester, O-methylphosphonate, and 5′-deoxy-5′-S-methylphosphonate linkages, we O-methylphosphonate, and 50-deoxy-50-S-methylphosphonate linkages, we developed procedures whichdeveloped allowed procedures the synthesis which of Hallowed-phosphinate the synthesis internucleotide of H-phosphinate linkage 7 (Figure internucleotide1). This linkage linkage could 7 be(Figure oxidized, 1). This sulfurized, linkage orcou amidatedld be oxidized, to form sulfurized, the O-methylphosphonate or amidated to form8a, O the-methylphosphonothioate O-methylphosphonate 9a8a,, O--methylphosphonamidatemethylphosphonothioate 9a10, ,OS--methylphosphonatemethylphosphonamidate8b and10, SS-methylphosphonate-methylphosphonothioate 8b and9b S- internucleotidemethylphosphonothioate linkages, respectively.9b internucleotide The insertion linkages, of the respectively. oxidation step The afterinsertion each ofH -phosphonate the oxidation couplingstep after step each [11 H,12-phosphonate] allowed us to coupling combine step easily [11 phosphonate-derived,12] allowed us to combine units with easily the phosphodiester phosphonate- onesderived and units to prepare with the unique phosphodiester set of modified ones oligonucleotides. and to prepare unique set of modified oligonucleotides.

FigureFigure 1. 1Overview. Overview of of possible possible combinations combinations of of the the phosphodiester phosphodiester3, 34,, 46, 6and and phosphonate phosphonate8a 8a, 8b, 8b, 9a, , 9b and 10 linkages. 9a, 9b and 10 linkages. 2. Results and Discussion 2. Results and Discussion 2.1. Study on Model Dimers 2.1. Study on Model Dimers Our results on novel nucleoside-O-methyl-(H)-phosphinates [2,3] as monomers for H-phosphonate chemistryOur results prompted on usnovel to develop nucleoside robust-O-methyl synthetic-(H protocols)-phosphinates that would[2,3] allowas monomers the synthesis for H of- oligonucleotidesphosphonate chemistry bearing anyprompted combination us to of develop phosphodiester robust synthetic3, phosphorothioate protocols that4, phosphoramidatewould allow the 6synthesis, O-methylphosphonate of oligonucleotides8a , bearingO-methylphosphonothioate any combination of phosphodiester9a, O-methyl-phosphonamidate 3, phosphorothioate10 4, Sphosphoramidate-methylphosphonate 6, 8bO-andmethylphosphonateS-methylphosphonothioate 8a, O-methylphosphonothioate9b internucleotide linkages 9a, (FigureO-methyl1). - Thephosphonamidate optimization of10, the S-methylphosphonate oxidative couplings 8b affording and S-methylphosphonothioate the abovementioned modified 9b internucleotide bonds was performedlinkages (Fig onure a model1). The dimer. optimiza Thus,tion of TentaGel the oxidative modified coupling with 4-s Naffording-benzoyl-2 the0-deoxy-3 abovementioned0-O-DMTr- cytidine-5modified 0bonds-hemisuccinate was performed11 was extendedon a model with dimer. thymidine- Thus,O T-methyl-(entaGelH modified)-phosphinate with or4-N thymidine-5-benzoyl-2′0- deoxy-5deoxy-3′0 -SO-methyl-(-DMTr-cytidineH)-phosphinate-5′-hemisuccinate monomer 112 towas provide extended TentaGel-attached with thymidine (dCT)-O- dimermethyl with-(H)- Hphosphinate-phosphinate or internucleotidethymidine-5′-deoxy linkage-5′-S12b-methyl. This-(H) linkage-phosphinate was subjected monomer to 2 oxidativeto provide procedures TentaGel- (oxidationattached (dCT)/sulfurization dimer with/amidation) H-phosphinate under internucleotide various conditions linkage (Scheme 12b. This1). linka All experimentsge was subjected with to Hoxidative-phosphinate procedures monomers (oxidation/sulfurization/amidation)2 were always compared to the under standard various thymidine conditionsH-phosphonate (Scheme 1).1 Allas aexperiments control of the with efficacy H-phosphinate of the reaction monomers conditions 2 were (For morealways details compared see Supporting to the standard Information). thymidine H- phosphonate 1 as a control of the efficacy of the reaction conditions (For more details see Supporting Information).

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Scheme 1 1.. Model dimer for the optimization of H--phosphonatephosphonate chemistry chemistry..

2.1.1. H--PhosphonatePhosphonate Coupling The optimization of of coupli couplingng reaction reaction of of HH-phosphinate-phosphinate monomers monomers 2 [2,32 [2] ,was3] was performed performed at 0.1 at 0.1M concentration M concentration in in acetonitrile acetonitrile-pyridine-pyridine mixture mixture (1:1 (1:1)) with with a a series series of of activating activating agents agents [[13]13] commonly used used in inH -phosphonateH-phosphonate chemistry chemistry such such as pivaloyl as pivaloyl chloride, chloride, adamantanecarbonyl adamantanecarbonyl chloride, 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinanechloride, 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (DMOCP), 2-oxide (DMOCP), diphenyl chloro-phosphatediphenyl chloro- (DPCP),phosphate and (DPCP) bis(2-oxo-3-oxazolidinyl)phosphinic, and bis(2-oxo-3-oxazolidinyl)phosphinic chloride chloride (OXP) (OXP) at 0.3 at M 0.3 concentration M concentration in acetonitrile-pyridinein acetonitrile-pyridine mixtures mixture (95:5).s (95:5). Of Of all all tested tested coupling coupling agents,agents, wewe selectedselected DMOCPDMOCP as the activator of choice for thethe condensationcondensation ofof bothboth HH-phosphonate-phosphonate 1 andand HH-phosphinates-phosphinates 2.. The only didifferencefference was the the durationduration ofof thethe coupling coupling step. step. The H-phosphinate-phosphinate monomers required 10 min condensation time compared to 55 minmin HH-phosphonate-phosphonate condensation.condensation. 2.1.2. Oxidation/Amidation/Sulfurization of H-Phosphinate Internucleotide Linkage 2.1.2. Oxidation/Amidation/Sulfurization of H-Phosphinate Internucleotide Linkage As we published previously [2,3], the use of water-containing oxidation mixtures such as As we published previously [2,3], the use of water-containing oxidation mixtures such as 0.1 M 0.1 M iodine in pyridine-water mixture (98:2) reported for the oxidation of H-phosphonate linkage iodine in pyridine-water mixture (98:2) reported for the oxidation of H-phosphonate linkage 12a [7,8] 12a [7,8] could not be used for the oxidation of H-phosphinate bond 12b due to its lability under could not be used for the oxidation of H-phosphinate bond 12b due to its lability under aqueous aqueous conditions and very fast hydrolytic cleavage. The use of 0.1 M iodine in pyridine-methanol conditions and very fast hydrolytic cleavage. The use of 0.1 M iodine in pyridine-methanol (50:50) MeO-P(V) (50:50) mixture led to only 80% yield of the expected(V) product. However, Atherton-Todd mixture led to only 80% yield of the expected MeO-P product. However, Atherton-Todd reaction(V) reaction [14–16] conditions (CCl4-pyridine-methanol) provided an acceptable 95% yield of MeO-P [14–16] conditions (CCl4-pyridine-methanol) provided an acceptable 95% yield of MeO-P(V) product. product. Moreover, no hydrolysis of internucleotide linkage was observed. Moreover, no hydrolysis of internucleotide linkage was observed. Our further research focused on the composition of the CCl4-based oxidation mixture, especially Our further research focused on the composition of the CCl4-based oxidation mixture, especially with respect to the base used and its basicity/nucleophilicity. The replacement of pyridine with with respect to the base used and its basicity/nucleophilicity. The replacement of pyridine with 1- 1-methylimidazole or 1-methylimidazole-triethylamine mixture, led to a quantitative oxidation of both methylimidazole or 1-methylimidazole-triethylamine mixture, led to a quantitative oxidation of both H-phosphonate and H-phosphinate linkages. We found that methanol and 3-hydroxypropionitrile H-phosphonate and H-phosphinate linkages. We found that methanol and 3-hydroxypropionitrile exhibited similar reactivity to provide methyl and 2-cyanoethyl esters, respectively, however, exhibited similar reactivity to provide methyl and 2-cyanoethyl esters, respectively, however, the 2- the 2-cyanoethyl ester represented more stable ester group. Since 3-hydroxypropionitrile is immiscible cyanoethyl ester represented more stable ester group. Since 3-hydroxypropionitrile is immiscible with tetrachloromethane, DCM was used as a co-solvent in oxidation mixture (Table1). with tetrachloromethane, DCM was used as a co-solvent in oxidation mixture (Table 1). Encouraged by these results and based on the fact that the amines were commonly used nucleophiles Encouraged by these results and based on the fact that the amines were commonly used in Atherton-Todd reaction [15,16], we examined the preparation of electroneutral oligonucleotides with nucleophiles in Atherton-Todd reaction [15,16], we examined the preparation of electroneutral non-bridging P–N bond using oxidative amidation of the H-phosphinate linkage. In contrast to the oligonucleotides with non-bridging P–N bond using oxidative amidation of the H-phosphinate reported phosphoramidate oligonucleotides [14,17,18], the phosphonamidate oligonucleotides have linkage. In contrast to the reported phosphoramidate oligonucleotides [14,17,18], the not been published so far. Two representative amines, the primary N,N-dimethylethylenediamine phosphonamidate oligonucleotides have not been published so far. Two representative amines, the which was already incorporated into phosphoramidate oligonucleotides [17,18] and morpholine [19] as primary N,N-dimethylethylenediamine which was already incorporated into phosphoramidate a representative of secondary amines were selected as amidation reagent. We examined various ratios of oligonucleotides [17,18] and morpholine [19] as a representative of secondary amines were selected tetrachloromethane and the amines in the mixture, and the time of the amidation reaction. A quantitative as amidation reagent. We examined various ratios of tetrachloromethane and the amines in the amidation of both H-phosphonate and H-phosphinate linkages was achieved after 180 min using a mixture mixture, and the time of the amidation reaction. A quantitative amidation of both H-phosphonate of CCl4/amine/DCM in 3:2:5 ratio (DCM was used to keep forming amine hydrochloride in the solution). and H-phosphinate linkages was achieved after 180 min using a mixture of CCl4/amine/DCM in 3:2:5 ratio (DCM was used to keep forming amine hydrochloride in the solution).

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The sulfurization of H-phosphinate 12b linkage to afford phosphonothioate bond was achieved using two methods. The first method employed elemental sulfur in various solvents and in the presence of base [9], and provided directly expected charged dimers. The reaction performed with 0.5 M solution of elemental sulfur in pyridine provided the desired products quantitatively within 20 min. Since this type of sulfurization could be only used as the last step of the synthetic cycles, we also focused on the second method employing sulfur-transfer reagent (N-[(2-cyanoethyl)-sulfanyl]succinimide, CSS) [20,21]. This method was applicable either in the last H-phosphonate cycle or after each synthetic cycle affording S-(2-cyanoethyl) esters which were then cleaved by β-elimination to provide phosphonothioate linkages. Thus the sulfurization performed with 0.2 M CSS in a mixture of acetonitrile and silylating agent BSTFA [20] (29:1) provided the S-(2-cyanoethyl)-protected dimers quantitatively within 30 min. β-Elimination reaction was performed at the end of the synthesis with 1 M DBU in acetonitrile for 3 min. The optimal reaction conditions of the individual oxidation steps are summarized in Table1.

Table 1. The conditions of oxidation, sulfurization and amidation.

Oxidation Time (min) Code Mixture H-Phosphonate H-Phosphinate

Ox-A CCl4/MeOH/MeIm (7:2:1) 15 30 Ox-B CCl4/MeOH/MeIm/Et3N (7:2:0.5:0.5) 10 20 0.1 M HOCH CH CN in Ox-C 2 2 15 30 CCl4/DCM/MeIm (6:2:2) Ox-D 0.5 M sulfur in pyridine 20 20 Ox-E 0.2 M CSS in ACN/BSTFA (29:1) 30 30 Ox-F CCl4/amine */DCM (3:2:5) 180 180 * Morpholine or N,N-dimethylethylendiamine.

2.1.3. Stability of Modified Internucleotide Linkages During the optimization of the condensation and oxidation steps, we observed different behavior and reactivity of both H-phosphonate and H-phosphinate internucleotide linkages. Therefore we decided to examine the stability of these linkages using a series of modified d(CT) dimers. All experiments were performed on LCMS at several time intervals. The stability of phosphorothioate and phosphonothioate linkages was examined in the presence of two potential desulfurizing agents—sodium periodate (0.05 M aqueous solution) and iodine (0.05 M solution in pyridine-water mixture (98:2)). It was reported that the periodate anion partially desulfurized the phosphorothioate TpT dimer [22]. In agreement with this, the treatment of phosphorothioate d(CT) dimer 14a with sodium periodate quantitatively afforded the product of desulfurization, d(CT) phosphate dimer 15a, after 12 h. In case of phosphonothioate dimer 14b, complete cleavage of the internucleotide linkage to phosphonate 16b was observed. On the other hand, S-methylthiophosphonate dimer 14c was cleaved to phosphonate 16c only partially (53%). The rest of the dimer underwent desulfurization and oxidation of S-methylphosphonate linkage to S(=O)-methylphosphonate 15d (47%). In case of iodine-promoted desulfurization, all tested dimers 14a–c were quantitatively and cleanly transformed to phosphate/phosphonate dimers 15a–c within 5 min of treatment. Neither cleavage nor oxidation of S-methylphosphonate in the case of dimer 14c was observed (Scheme2). We also tested the stability of P–N bonds of dimers 17e–h under acidic conditions (pH = 1 for 5 h; Scheme2). Comparing the stability of phosphoramidate and phosphonamidate linkages, phosphoromorpholidate dimer 17e exhibited higher stability (35% hydrolysis to dimer 15a) than phosphonomorpholidate dimer 17f (78% hydrolysis to dimer 15b). Suprisingly, S-methyl- phosphonomorpholidate dimer 17g showed the highest stability, with only 18% hydrolysis to dimer 15c. Molecules 2019, 24, x FOR PEER REVIEW 4 of 11

The sulfurization of H-phosphinate 12b linkage to afford phosphonothioate bond was achieved using two methods. The first method employed elemental sulfur in various solvents and in the presence of base [9], and provided directly expected charged dimers. The reaction performed with 0.5 M solution of elemental sulfur in pyridine provided the desired products quantitatively within 20 min. Since this type of sulfurization could be only used as the last step of the synthetic cycles, we also focused on the second method employing sulfur-transfer reagent (N-[(2-cyanoethyl)- sulfanyl]succinimide, CSS) [20,21]. This method was applicable either in the last H-phosphonate cycle or after each synthetic cycle affording S-(2-cyanoethyl) esters which were then cleaved by β- elimination to provide phosphonothioate linkages. Thus the sulfurization performed with 0.2 M CSS in a mixture of acetonitrile and silylating agent BSTFA[20] (29:1) provided the S-(2-cyanoethyl)- protected dimers quantitatively within 30 min. β-Elimination reaction was performed at the end of the synthesis with 1 M DBU in acetonitrile for 3 min. The optimal reaction conditions of the individual oxidation steps are summarized in Table 1.

Table 1. The conditions of oxidation, sulfurization and amidation.

Oxidation Time (min) Code Mixture H-Phosphonate H-Phosphinate Ox-A CCl4/MeOH/MeIm (7:2:1) 15 30 Ox-B CCl4/MeOH/MeIm/Et3N (7:2:0.5:0.5) 10 20 0.1 M HOCH2CH2CN in Ox-C 15 30 CCl4/DCM/MeIm (6:2:2) Ox-D 0.5 M sulfur in pyridine 20 20 Ox-E 0.2 M CSS in ACN/BSTFA (29:1) 30 30 Ox-F CCl4/amine */DCM (3:2:5) 180 180 * Morpholine or N,N-dimethylethylendiamine.

2.1.3. Stability of Modified Internucleotide Linkages During the optimization of the condensation and oxidation steps, we observed different behavior and reactivity of both H-phosphonate and H-phosphinate internucleotide linkages. Therefore we decided to examine the stability of these linkages using a series of modified d(CT) dimers. All experiments were performed on LCMS at several time intervals. The stability of phosphorothioate and phosphonothioate linkages was examined in the presence of twoMolecules potential2019 ,desulfurizing24, 1872 agents—sodium periodate (0.05 M aqueous solution) and iodine (0.055 of 12 M solution in pyridine-water mixture (98:2)).

Molecules 2019, 24, x FOR PEER REVIEW 5 of 11 Molecules 2019, 24, x FOR PEER REVIEW 5 of 11 Molecules 2019, 24, x FOR PEER REVIEW 5 of 11 phosphonate 16b was observed. On the other hand, S-methylthiophosphonate dimer 14c was cleaved phosphonate 16b was observed. On the other hand, S-methylthiophosphonate dimer 14c was cleaved to phosphonate 16c only partially (53%). The rest of the dimer underwent desulfurization and tophosphonate phosphonate 16b 16c was only observed partially. On the (53%). other The hand, rest S - ofmethylthiophosphonate the dimer underwent dimer desulfurization 14c was cleaved and oxidation of S-methylphosphonate linkage to S(=O)-methylphosphonate 15d (47%). oxidationto phosphonate of S-methylphosphonate 16c only partially linkage (53%). to The S(=O) rest-methylphosphonate of the dimer underwent 15d (47%). desulfurization and In case of iodine-promoted desulfurization, all tested dimers 14a–c were quantitatively and oxidationIn case of ofS- methylphosphonate iodine-promoted desulfurization, linkage to S(=O) all-methylphosphonate tested dimers 14a– 15dc were (47%). quantitatively and cleanly transformed to phosphate/phosphonate dimers 15a–c within 5 min of treatment. Neither cleanlyIn transformed case of iodine to- promoted phosphate/phosphonate desulfurization, dimers all tested 15a – dimersc within 14a 5 – minc were of treatment. quantitatively Neither and cleavage nor oxidation of S-methylphosphonate in the case of dimer 14c was observed (Scheme 2). cleavcleanlyage transformednor oxidation to of phosphate/phosphonate S-methylphosphonate in dimers the case 15a of– dimerc within 14c 5was min observed of treatment. (Scheme Neither 2). We also tested the stability of P–N bonds of dimers 17e–h under acidic conditions (pH = 1 for 5 cleavWeage also nor testedoxidation the stabilityof S-methylphosphonate of P–N bonds of indimers the case 17e of–h dimer under 14c acidic was conditions observed ((pHScheme = 1 for 2). 5 h; Scheme 2). Comparing the stability of phosphoramidate and phosphonamidate linkages, h; SchemeWe also 2 )tested. Comparin the stabilityg the of stability P–N bonds of phosphoramidateof dimers 17e–h under and acidic phosphon conditionsamidate (pH linkages, = 1 for 5 Schemephosphoromorpholidate 2. Stability of phos dimerphorothioate/phosphonothioate 17e exhibited higher stability and phosphoramidate/phosphonamidate (35% hydrolysis to dimer 15a) than phhosphoromorpholidate; SchemeScheme 2.2).Stability Comparin of dimer phosphorothioateg the 17e stability exhibited of/phosphonothioate higher phosphoramidate stability ( and35 % andphosphoramidate hydrolysis phosphon toamidate dimer/phosphonamidate 15a linkages,) than internucleotidephosphonomorpholidate linkages. dimer 17f (78% hydrolysis to dimer 15b). Suprisingly, S-methyl- phosphonomorpholidatephosphoromorpholidateinternucleotide linkages. dimerdimer 17e17f exhibited (78% hydrolysis higher stability to dimer (35% 15hydrolysisb). Suprisingly, to dimer S15a-methyl) than- phosphonomorpholidate dimer 17g showed the highest stability, with only 18% hydrolysis to dimer phosphonophosphonomorpholidatemorpholidate dimerdimer 17g 17f showed (78% the hydrolysis highest stabilit to dimery, with 15onlyb). 18% Suprisingly, hydrolysis S to-methyl dimer- It15 wasc. reported that the periodate anion partially desulfurized the phosphorothioate TpT dimer 15phosphonoc. Moreover,morpholidate phosphonamidate dimer 17g linkagesshowed the derived highest from stabilit primaryy, with aminesonly 18% were hydrolysis much moreto dimer stable than [22]. In agreementMoreover, with phosphonamidate this, the treatment linkages derived of phosphorothioate from primary aminesd(CT) were dimer much 14a more with stable sodium the15c linkages. Moreover, derived phosphonamidate from secondary linkages amines derived (Scheme from2 ). primary Dimethylethylendiamino-phosphonamidate amines were much more stable periodatethan quantitatively the linkages afforded derived fromthe product secondary of desulfurization amines (Scheme, d(CT) 2). Dphosphateimethylethylendiamin dimer 15a,o -after dimerthan Moreover, the17i and linkages phosphonamidate dimethylethylendiamino- derived from linkages secondary derivedS-methylphosphonamidate amines from primary(Scheme amines 2). D imethylet were dimer muchhylendiamin17j moreexhibited stableo- similar phosphonamidate dimer 17i and dimethylethylendiamino-S-methylphosphonamidate dimer 17j 12 h.stability, phosphonamidateInthan case the withof linkagesphosphonothioate only dimer 6% derived hydrolysis 17i and from dimer dimethylethylend to secondary the 14b dimers, complete amines15biamin andc leavageo (Scheme-S15c-methylphosphonamidate, respectively. of 2 ).the D internucleotideimethylet Dimethylethylendiamino-hylendiamin dimer linkage 17oj- to exhibited similar stability, with only 6% hydrolysis to the dimers 15b and 15c, respectively. exhibitedphosphonamidate similar stability dimer ,17 withi and only dimethylethylend 6% hydrolysisiamin to theo-S -methylphosphonamidatedimers 15b and 15c, respectively dimer 17.j phosphoramidateDimethylethylendiamino dimer -17hphosphoramidatewas hydrolyzed dimer to the17h same was extenthydrolyzed as its to the same extent derivative as its 17e (36% Dimethylethylendiaminoexhibited similar stability-phosphoramidate, with only 6% dimer hydrolysis 17h was to the hydrolyzed dimers 15 tob theand same 15c, extent respectively as its . hydrolysismorpholino to derivative dimer 15a 17e). (36% hydrolysis to dimer 15a). morpholinoDimethylethylendiamino derivative 17e-phosphoramidate (36% hydrolysis to dimer dimer 17h15a ).was hydrolyzed to the same extent as its morpholino derivative 17e (36% hydrolysis to dimer 15a). 2.2.2.2. Synthetic Synthetic Protocols for for Oligonucleotide Oligonucleotide Synthesis Synthesis 2.2. Synthetic Protocols for Oligonucleotide Synthesis 2.2. BasedSyntheticBased on Protocols the results results for Oligonucleotide obtained obtained on onSynthesis the the level level of dimers, of dimers, we have we havedeveloped developed three synthetic three synthetic Based on the results obtained on the level of dimers, we have developed three synthetic protocolsprotocols (Synthetic (Synthetic protocols protocols A, A, B, and B, andC), and C), tested and their tested e ffi thecacyir efficacy on model on homo-oligothymidylates model homo- protocolsBased (Synthetic on the results protocols obtained A, on B, theand level C), of and dimers, tested we the haveir efficacy developed on threemodel synthetic homo- dTol10igothymidylates1–dT107 modified dT101 with–dT107 various modified internucleotide with various internucleotide linkages (Table linkages2). (Table 2). olprotocolsigothymidylates (Synthetic dT 101 protocols–dT107 modified A, B, with and various C), and internucleotide tested their linkages efficacy (Table on 2 model). homo- oligothymidylates dT101–dT107 modified with various internucleotide linkages (Table 2). Table 2. Summary of model homo-oligothymidylates dT101–dT107. TableTable 2. 2. SummarySummary of model of model homo homo-oligothymidylates-oligothymidylates dT101–dT101071. –dT107. Table 2. Summary of model homo-oligothymidylatesSynthetic dT10 1–dT107. SyntheticSynthetic ProtocolMALDI TOF Calcd InternucleotideInternucleotide linkages Linkages Code Code SequenceSequence 5′ 5→3′3 Protocol (HPLC MALDIMALDI TOF TOFCalcd Calcd Found Internucleotide linkages Code Sequence 5′→0 3′ 0 ProtocolSynthetic(HPLC (HPLC Purity) Found → Purity) MALDIFound TOF Calcd Internucleotide linkages Code Sequence 5′→3′ ProtocolPurity (HPLC) A (70%), B (74%), Found dT 1 d(T TTTTTTTTT) A Purity(70%),) B 3020.5516 3020.5733 dT10101 d(T T T T T T T T T T) A (70%),C (64%) B 3020.5516 3020.5733 dT101 d(T T T T T T T T T T) (74%), C (64%) 3020.5516 3020.5733 (74A%), (7 C0%), (64 B%) dT101 d(T T T T T T T T T T) 3020.5516 3020.5733 (74%), C (64%) dTdT10102 2 d(Td(T T T T TT TT TT TT TT T)T ) A (A59(59%),%), B (77B%)(77%) 3164.3460 3164.3460 3164.32193164.3219 dT102 d(T T T T T T T T T T) A (59%), B (77%) 3164.3460 3164.3219 dT102 d(T T T T T T T T T T) A (59%), B (77%) 3164.3460 3164.3219 dTdT10103 3 d(Td(T T TTTT T TT TTT T TT TT)B T) B (36(36%),%), C (88C%)(88%) 3187.30 3187.304949 3187.17033187.1703 dT103 d(T T T T T T T T T T) B (36%), C (88%) 3187.3049 3187.1703 dT103 d(T T T T T T T T T T) B (36%), C (88%) 3187.3049 3187.1703 dT104 d(T T T T T T T T T T) B (42%), C (70%) 3229.3859 3229.1063 dTdT10410 4 d(T T T T TTT T TT T TTT TT T)T) B (42B %),(42%), C (70C%)(70%) 3229.3859 3229.3859 3229.1063 3229.1063

dT1054 d(T T T T T T T T T T) B (42C%), (30%) C (70 %) 3070.3229.32479859 3070.73293229.1063 dT105 d(T T T T T T T T T T) C (30%) 3070.2479 3070.7329 dT105 d(T TT TTT TTT T) C (30%) 3070.2479 3070.7329 dT1065 d(T T T T T T T T T T) C (75%)(30%) 2979.983070.247939 2980.39703070.7329 dT106 d(T T T T T T T T T T) C (75%) 2979.9839 2980.3970

dTdT101076 6 d(Td(T T T T T T T T T T T T TT T) T) C (77%)(75%) C (75%) 3028.2979.981696 2979.983939 3028.40482980.39702980.3970 dT107 d(T T T T T T T T T T) C (77%) 3028.1696 3028.4048

dT107 d(T T T T T T T T T T) C (77%) 3028.1696 3028.4048 dT107 d(T T T TTT TTT T) C (77%) 3028.1696 3028.4048 2.2.1. Synthetic Protocol A 2.2.1. Synthetic Protocol A 2.2.1.This Synthetic protocol Protocol was A used for the synthesis of oligonucleotides fully modified with either 2.2.1.This Synthetic protocol Protocol was used A for the synthesis of oligonucleotides fully modified with either phosphonate/phosphate 18 or thiophosphonate/ 19 linkages (Scheme 3, Table 2). The phosphonate/phosphateThis protocol was 18 used or thiop for hosphonate/thiophosphate the synthesis of oligonucleotides 19 linkages fully (Scheme modified 3, Table with 2 ). either The nucleosideThis protocol H-phosphonates was used 1 andfor O-methyl the synthesis-(H)-phosphinates of oligonucleotides 2 were coupled on fully solid modified support using with either nucleosidephosphonate/phosphate H-phosphonates 18 1or and thiop O-hosphonate/thiophosphatemethyl-(H)-phosphinates 2 were19 linkages coupled (Scheme on solid 3 support, Table 2 using). The H-phosphonate chemistry, to provide oligonucleotide 20. Since the use of H-phosphonate capping phosphonateHnucleoside-phosphonate H-/phosphonatesphosphate chemistry,18 to 1 orprovideand thiophosphonate O-methyl oligonucleotide-(H)-phosphinates/thiophosphate 20. Since 2 werethe use 19coupled linkagesof H- phosphonateon so (Schemelid support capping3, Tableusing 2). The [23] led, in our hands, to the formation of substantial amount of side products, this step was omitted. nucleoside[23]H-phosphonate led, in ourH-phosphonates hands, chemistry to the, toformation 1provideand O -methyl-( ofoligonucleotide substantialH)-phosphinates amount 20. Since of side the 2products, usewere of coupledH -thisphosphonate step on was solid omitted. capping support using After last synthetic cycle, all internucleotide linkages were either oxidized or sulfurized to afford HAfter[23]-phosphonate led last, in synthetic our hands, chemistry, cycle, to t heall to formation internuc provideleotide of oligonucleotide substantial linkages amount were20. either Sinceof side oxidized the products, use ofor thisH sulfurized-phosphonate step was to omitted. afford capping [23] oligonucleotides 21 or 19, respectively (Scheme 3). The use of non-aqueous oxidation mixtures (Table led,oligonucleotideAfter in last our synthetic hands,s 21 or tocycle, 19 the, respectively all formation internuc (leotideScheme of substantial linkages 3). The use were amount of neitheron- ofaqueous oxidized side products,oxidation or sulfurized mixtures this step to (Tableafford was omitted. 1; Ox-A, Ox-B, and Ox-C) that oxidized P–H bonds to P–OR bond (R = CH3, 2-cyanoethyl) led to 1oligonucleotide; Ox-A, Ox-B, ands 21 Oxor 19-C,) respectivelythat oxidized (Scheme P–H bonds 3). The to use P– ORof n onbond-aqueous (R = CH oxidation3, 2-cyanoethyl mixtures) led(Table to Aftercomplete last syntheticoxidation. cycle,The methyl all internucleotide and 2-cyanoethyl linkages ester were groups either were oxidized easily removedor sulfurized using to afford complete1; Ox-A, Oxoxidation-B, and . OxThe-C ) methylthat oxidized and 2P-cyanoethyl–H bonds to ester P–OR groups bond (R were = CH easily3, 2-cyanoethyl removed ) usingled to oligonucleotidesthiophenol/TEA/DMF21 or and19, respectivelygaseous ammonia, (Scheme respectively3). The use, to ofafford non-aqueous the final product oxidation 18. mixtures (Table1; thiophenolcomplete /TEA/DMFoxidation. andThe gaseous methyl ammonia, and 2-cyanoethyl respectively ester, to afford groups the were final easilyproduct removed 18. using thiophenol/TEA/DMF and gaseous ammonia, respectively, to afford the final product 18.

Molecules 2019, 24, 1872 6 of 12

Ox-A, Ox-B, and Ox-C) that oxidized P–H bonds to P–OR bond (R = CH3, 2-cyanoethyl) led to complete oxidation. The methyl and 2-cyanoethyl ester groups were easily removed using thiophenol/TEA/DMF Molecules 2019, 24, x FOR PEER REVIEW 6 of 11 Moleculesand gaseous 2019, 24 ammonia,, x FOR PEER respectively, REVIEW to afford the final product 18. 6 of 11

Scheme 3 3.. SyntheticSynthetic prot protocolocol A A—synthesis—synthesis of fully modified modified strands strands..

The sulfurization of of the the HH--phosphonatephosphonate/H/H-phosphinate-phosphinate linkages linkages proceeded proceeded smoothly smoothly using using a solutiona solution of of elemental elemental sulfur sulfur in in pyridine pyridine (Table (Table 1;; OxOx-D-D).). It It is is important important to to note note that that the the elimination elimination of traces of of water water from from the the oxidation oxidation and sulfurization and sulfurization mixtures mixtures was of the was utmost of the importance. utmost importance. Therefore, Therefore,all the oxidation all the mixturesoxidation were mixtures dried were over dried 3 Å molecular over 3 Å mo sieveslecular for sieves 16 h prior for 16 the h use.prior This the use. protocol This (Forprotocol more (For details more see details Materials see and Materials Methods, and Supporting Methods, Information) Supporting wasInformation successfully) was applied successfully for the applied for the preparation of oligonucleotides dT101, dT102, ON-8, and ON-9 (Tables 2 and 3). preparationapplied for the of oligonucleotidespreparation of oligonucleotidesdT101, dT102, ON-8 dT10,1 and, dT10ON-92, ON(Tables-8, and2 ONand-39). (Tables 2 and 3). 2.2.2. Synthetic Protocol B 2.2.2. Synthetic Protocol B This protocol combined phosphoramidite and H-phosphonate chemistries to introduce This protocol combined phosphoramidite and H-phosphonate chemistries to introduce the the phosphodiester/phosphorothioate and O-methylphosphonate/O-methylphosphonothioate phosphodiester/phosphorothioate and O-methylphosphonate/O-methylphosphonothioate inter- inter-nucleotide linkages, respectively, at specific positions of the oligonucleotide strand (Scheme4). nucleotide linkages, respectively, at specific positions of the oligonucleotide strand (Scheme 4). The TheIII PIII triester bonds were oxidized with 1M t-BuOOH in DCM or sulfurized using Sulfurizing PIII triester bonds were oxidized with 1M t-BuOOH in DCM or sulfurized using Sulfurizing Reagent Reagent II (DDTT, Glen Research, Sterling, VA, USA) after each phosphoramidite coupling step. The II (DDTT, Glen Research, Sterling, VA, USA) after each phosphoramidite coupling step. The oxidation of the H-phosphinate internucleotide linkages in oligonucleotide 22 was performed after each oxidation of the H-phosphinate internucleotide linkages in oligonucleotide 22 was performed after step with the mixture Ox-C utilizing 3-hydroxypropionitrile (Table1) to a fford product 23. The use of each step with the mixture Ox-C utilizing 3-hydroxypropionitrile (Table 1) to afford product 23. The the oxidation mixtures Ox-A and Ox-B containing methanol did not afford the desired oligonucleotide. use of the oxidation mixtures Ox-A and Ox-B containing methanol did not afford the desired The explanation for this behaviour might lie in the facts that neutral methyl esters of phosphonates oligonucleotide. The explanation for this behaviour might lie in the facts that neutral methyl esters of could serve as powerful methylating agents in the presence of a nucleophile such as sulfur compound phosphonates could serve as powerful methylating agents in the presence of a nucleophile such as or pyridine, and that the cleavage of the methyl protecting group of the phosphonate moiety gave sulfur compound or pyridine, and that the cleavage of the methyl protecting group of the rise to a P–OH reactive center that interfered with the coupling steps leading to a complex mixture phosphonate moiety gave rise to a P–OH reactive center that interfered with the coupling steps of products. leading to a complex mixture of products.

Scheme 4. Synthetic protocol B—synthesis of oligonucleotides modified at specific positions. Scheme 4. Synthetic protocol B—synthesis of oligonucleotides modified at specific positions. The synthesis of S-protected phosphonothioate internucleotide linkages in oligonucleotide 24 was The synthesis of S-protected phosphonothioate internucleotide linkages in oligonucleotide 24 achieved using the sulfur-transfer reagent (CSS) [20,21] (Table1; Ox-E) to form S-(2-cyanoethyl) ester was achieved using the sulfur-transfer reagent (CSS) [20,21] (Table 1; Ox-E) to form S-(2-cyanoethyl) of the phosphonothioate linkage. After the synthesis of the entire oligonucleotide, the S-(2-cyanoethyl) ester of the phosphonothioate linkage. After the synthesis of the entire oligonucleotide, the S-(2- cyanoethyl) ester groups were removed by β-elimination using 1 M DBU in acetonitrile. Surprisingly, gaseous ammonia could not be used for the β-elimination reaction since it caused cleavage of protected phosphonothioate internucleotide linkages to afford the degradation of the oligonucleotide strand. The introduction of oxidation or sulfurization after each coupling step allowed the use of standard acetic anhydride-based capping procedure. The protocol (For more details see Materials

Molecules 2019, 24, 1872 7 of 12 ester groups were removed by β-elimination using 1 M DBU in acetonitrile. Surprisingly, gaseous ammonia could not be used for the β-elimination reaction since it caused cleavage of protected phosphonothioateMolecules 2019, 24, x FOR internucleotide PEER REVIEW linkages to afford the degradation of the oligonucleotide strand.7 of 11 The introduction of oxidation or sulfurization after each coupling step allowed the use of standard aceticand Methods,anhydride-based Suppor cappingting Information procedure.) The was protocol successfully (For more applied details see for Materials the preparation and Methods, of oligonucleotides dT101, dT102, dT103, dT104, ON-4, ON-5, ON-6, ON-7, ON-10, and ON-11 (Tables 2 Supporting Information) was successfully applied for the preparation of oligonucleotides dT101, dT102, and 3). dT103, dT104, ON-4, ON-5, ON-6, ON-7, ON-10, and ON-11 (Tables2 and3).

2.2.3. Synthetic Protocol C This protocolprotocol combined combined phosphoramidite phosphoramidite and HH-phosphonate-phosphonate chemistries,chemistries, and allowed allowed the the introductionintroduction of of phosphoramidate phosphoramidate and phosphonamidateand phosphonamidate linkages linkages at specific at sites specific of the oligonucleotide sites of the 25oligonucleotide(Scheme5). We 25 found(Scheme that 5) the. WeH found-phosphonate that the andH-phosphonateO-methyl-( H and)-phosphinate O-methyl-(H internucleotide)-phosphinate linkagesinternucleotide in oligonucleotide linkages in oligonucleotide26 were neither 26 cleaved were neither nor oxidized cleaved by nor oxaziridine oxidized by CSO oxaziridine [24–26] which CSO represented[24–26] which a mild represented phosphoramidite a mild phosphoramidite oxidation agent. oxidation This reagent agent allowed. This reagent selective allowed oxidation selective of PIII linkageoxidation in of the PIII presence linkage ofin Hthe-phosphonate presence of H or-phosphonateH-phosphinate or H bonds.-phosphinate The amidation bonds. T ofhe the amidationP–H bond of inthe27 P–hasH bond to be in performed 27 has to atbe theperformed end of the at the oligonucleotide end of the oligonucleotide synthesis. This synthesis synthetic. T arrangementhis synthetic didarrangement not allow did the usenot ofallow acetic the anhydride-based use of acetic anhydride capping- duebased to thecapping instability due to of theH-phosphonate instability of and H- Hphosphonate-phosphinate and linkages H-phosphinat in 27. Thee linkages use of UniCap in 27. The [27 use] could of UniCap be a solution, [27] could however, be a solution, in our handshowever, we didin our not hands observe we any did improvement.not observe any improvement. The amidation o off PP–H–H bondsbonds with with morpholine morpholine or orN,NN-,dimethylenediamineN-dimethylenediamine in 27 in (Sche27 (Schememe 5) w5as) wasperformed performed using using CCl4 CCl/amine/DCM4/amine/DCM mixture mixture (Table (Table 1; Ox1; -Ox-FF). Such). Such mixed mixed sequence sequencess containing containing P- P-morpholinemorpholine andand P–P–OROR linkageslinkages cannot cannot be prepared be prepared using using protocol protocol B because B because each each detritylation detritylation step stepwas accompanied was accompanied with a withpartial a hydrolysis partial hydrolysis of the P-morpholine of the P-morpholine bond due tobond the residual due to water the residual content waterin detritylation content in mixture detritylation. Recently mixture., Damha and Recently, Vlaho [28] Damha reported and the Vlaho preparation [28] reported of a series the preparationphosphoramidate of a series oligonucleotides phosphoramidate via oxidative oligonucleotides amidation via using oxidative primary amidation amines ( usingN,N-dimethyl primary- aminesethylendiamine, (N,N-dimethyl-ethylendiamine, 3-(dimethylamino)propylami 3-(dimethylamino)propylamine,ne, 4-dimethylaminobutylamine, 4-dimethylaminobutylamine, isopentylamine) isopentylamine)which were completely which wereresistant completely to detritylation resistant conditions to detritylation. conditions. This protocol (For(For more more details details see see MaterialsMaterials and and Methods, Methods, SupportingSupporting Information) Information) waswas successfully applied applied for for prepar preparationation of of oligonucleotides oligonucleotides dTdT10310, dT3, dT10410, dT4, 10dT5, 10dT5,10dT6, dT10610, 7dT, ON107-,12ON-12, ON-, ON-1313, ON,-ON-1414, and, andON-ON-1515 (Table(Tabless 2 and2 and 3). 3).

Scheme 5.5. Synthetic protocol C C—synthesis—synthesis of phosphoramidatephosphoramidate/phosphonamidate/phosphonamidate strands. 2.2.4. Synthesis of Hetero-Oligonucleotides 2.2.4. Synthesis of Hetero-Oligonucleotides To verify general usefulness of the above mention protocols, we also prepared a set To verify general usefulness of the above mention protocols, we also prepared a set of hetero- of hetero-oligonucleotides modified at specific positions with phosphodiester/phosphonate, oligonucleotides modified at specific positions with phosphodiester/phosphonate, phosphorothioate/phosphonothioate and phosphoramidate/phosphonamidate linkages (Table3). These phosphorothioate/phosphonothioate and phosphoramidate/phosphonamidate linkages (Table 3). oligonucleotides were prepared in excellent yields and purity. Therefore, the developed protocols These oligonucleotides were prepared in excellent yields and purity. Therefore, the developed represent novel and effective approaches to the synthesis of variety of phosphonate oligonucleotides protocols represent novel and effective approaches to the synthesis of variety of phosphonate modified with diverse types of linkages (For more details, and hybridization properties of ON-1–ON-15 oligonucleotides modified with diverse types of linkages (For more details, and hybridization see Supporting Information). properties of ON-1–ON-15 see Supporting Information).

Molecules 2019, 24, 1872 8 of 12 Molecules 2019, 24, x FOR PEER REVIEW 8 of 11 Molecules 2019, 24, x FOR PEER REVIEW 8 of 11 Table 3. ON-1 ON-15 Molecules 2019, 24, x FOR PEERTable REVIEW 3Summary. Summary ofof hetero-oligonucleotideshetero-oligonucleotides ON-1––ON-15. . 8 of 11 Molecules 2019, 24, x FOR PEERTable REVIEW 3. Summary of hetero-oligonucleotides ON-1–ON-15. 8 of 11 Table 3. Summary of hetero-oligonucleotidesSyntheticSynthetic ON-1–ON -15. MALDIMALDI TOF TOF Internucleotide Linkages Code Sequence 5 3 Synthetic Internucleotide LinkagesTable 3Code. Summary ofSequence hetero-oligonucleotides 5′0→→3′0 ProtocolProtocol ON-1–ON -15. MALDI TOF Internucleotide Linkages Code Sequence 5′→3′ Protocol Calcd Found (HPLC(HPLCSynthetic Purity) Purity) CalcdMALDI TOFFound → (HPLCSynthetic Purity) Calcd Found Internucleotide Linkages ON-1ONCode-1 d(Gd(GSequence C C A ATA T A TCAT5′ C A3′ C) C) ProtocolBB (86%)(86%) 2729.43 2729.4322MALDI22 TOF 2729.5331 Internucleotide Linkages CodeON-1 d(GSequence C A T A 5′T →C A3′ C) (HPLCProtocolB (86%) Purity) 2729.43Calcd22 2729.5331Found ON-2ON-2 d(Gd(G C CAT A T ATA T C AAC C) ) BB (84%) 2761.3Calcd865 2761.1589Found ON-12 d(G C A T A T C A C)C) (HPLCB (86%)(84%)(84%) Purity) 2729.432761.3 2761.386586522 2761.15892729.5331 ON-1 d(G C A T A T C A C) B (86%) 2729.4322 2729.5331 ON-3ON-23 d(Gd(G C CA AT T AA T C AA CC) ) BB (84%)(81%)(81%) 2761.32809.3 2809.3180865180 2809.32922761.1589 ONON--23 d(Gd(G CC AA TT AA TT CC AA CC)) BB (84%)(81%) 2761.32809.3865180 2761.15892809.3292

ON-4ON-34 d(Gd(G C CA AT AT TCA TA CC)B A CC)) B (81%)(21%)(21%) 2809.32771.4 2771.4792187920 2771.39832809.3292 ON-4 d(G C A T A T C A C) B (21%) 2771.4792 2771.3983 ONON--35 d(Gd(G CC AA TT AA TT CC AA CC)) BB (81%)(56%) 2809.32899.2964180 2809.32922899.2376 ON-5ON-45 d(Gd(Gd(G C CA AT T AA T C AA C)CC) ) BB (21%)(56%)(56%) 2771.42899. 2899.29642964792 2899.23762771.3983 ON-6 d(G C A T A T C A C) B (51%) 2819.4106 2819.2887 ONON--456 d(Gd(d(GG CC AA TT AA TT CC AA C)CC)) BB (21%)(56%)(51%) 2771.42899.2819.41296479206 2771.39832899.23762819.2887 ON-6ON-7 d(Gd(G C CA AT TA A TCT C AA CC)) BB (43%)(51%) 2851.36 2819.410650 2819.28872851.3559 ONON--56 d(d(GG CC AA TT AA TT CC AA CC)) BB (56%)(51%) 2899.2819.41296406 2899.23762819.2887 ON-7 d(G C A T A T C A C) B (43%) 2851.3650 2851.3559 ONON-7ON--68 d(Gd(Gd(G C CC A AAT TT AA T TT CC AA C)CC) ) BAB (51%) (76%)(43%) 2819.412723.5 2851.365047706 2819.2887 2851.35592723.7076 ON-78 d(G C A T A T C A CC)) AB (43%)(76%) 2851.362723.547750 2851.35592723.7076 ON-8ON-9 d(Gd(G C CA AT AT TCA TA CC)A A C) A (84%)(76%) 2851.36 2723.547750 2723.70762851.2513 ONON--789 d(Gd(G CC AA TT AA TT CC AA CC)C)) BA (43%)(76%)(84%) 2851.362723.52851.364775500 2851.35592723.70762851.2513 ON-10 d(G C A T A T C A C) B (86%) 2771.4791 2771.3636 ONON-9ON--1089 d(Gd(Gd(G C CC A AA T T AA TT CC AAA C)CC)C) ) AABA (76%)(86%)(84%)(84%) 2723.52851.362771.4 2851.365047779150 2723.7076 2851.25132771.3636 ON-11 d(G C A T A T C A C) B (86%) 2803.4335 2803.5659 ON--10119 d(Gd(G CC AA TT AA TT CC AA CC)C)) AB (84%)(86%) 2851.362771.42803.437915350 2851.25132771.36362803.5659 ON-10 d(G CA TA TCA C) B (86%) 2771.4791 2771.3636 ONON--1012 d(Gd(G CC AA TT AA TT CC AA C)C) BC (86%)(59%) 2771.42930.7279113 2771.36362930.6797 ON-1112 d(G C A T A T C A CC)) BC (86%)(59%) 2803.432930.723513 2803.56592930.6797 ON-11ON-13 d(Gd(G C CA AT T AAT T C A CC) ) CB (66%)(86%) 2975.86 2803.433531 2803.56592975.7741 ONON--111213 d(Gd(G CC AA TT AA TT CC AA CC)) BC (86%)(59%)(66%) 2803.432930.722975.86351331 2803.56592930.67972975.7741 ON-14 d(G C A T A T C A C) C (78%) 2888.6743 2888.8365 ONON-12ON--121314 d(Gd(Gd(G C CCA AA T TA AA TTCT CC AA C)C)C) CCC (59%)(66%)(78%)(59%) 2930.722975.862888.67 2930.7213133143 2930.6797 2930.67972975.77412888.8365 ON-13 d(G C A T A T C A C) C (66%) 2975.8631 2975.7741 ON-13ON-1415 d(Gd(G C CA AT TA A TCT C AA C)C) CC (78%)(79%)(66%) 2888.672933.81 2975.86314362 2975.77412888.83652933.7591 ONON--1415 d(Gd(G CC AA TT AA TT CC AA C)C) CC (78%)(79%) 2888.672933.814362 2888.83652933.7591 ON-14 d(G CATATCAC)C (78%) 2888.6743 2888.8365 ON-15 d(G C A T A T C A C) C (79%) 2933.8162 2933.7591 ON-15 d(G C A T A T C A C) C (79%) 2933.8162 2933.7591 3. Materials and Methods ON-15 d(G CATATCAC)C (79%) 2933.8162 2933.7591 3. Materials and Methods 33.1. M Generalaterials Information and Methods 3.3 Materials3.1. M Generalaterials andInformation and Methods Methods 3.1 GeneralThe 5′ →Information3′ trityl-off synthesis of oligonucleotide was performed using phosphoramidite and H- The 5′→3′ trityl-off synthesis of oligonucleotide was performed using phosphoramidite and H- 3.1.3.1phosphonate General General Information Information method on GeneSyn and MOS synthesizers (IOCB Prague, Prague, Czech Republic) phosphonateThe 5′→3′ method trityl-off on synthesis GeneSyn of and oligonucleotide MOS synthesizer was performeds (IOCB Prague using, Praguephosphoramidite, Czech Republic) and H- using 3′-O-dimethoxytritylnucleoside-5′-hemisuccinate-modified LCAA-CPG and TentaGel solid phosphonateusingTheThe 3′ 5 -5′O→-3dimethoxytrit3′ method trityl-otrityl-off ffon synthesis ylnucleosideGeneSyn of ofand oligonucleotide oligonucleotide-5′ MOS-hemisuccinate synthesizer was was-modified performeds (IOCB performed Prague LCAA using using-,CPG phosphoramiditePrague phosphoramidite an,d C TentaGelzech Republic) and solid H and- supports0 →in a0 0.5 µmol scale. For the phosphoramidite chemistry all monomers, and oxidation and H-phosphonatephosphonateusingsupports 3′- Oin- dimethoxytrita method0.5 method µmol on onscale. GeneSynylnucleoside GeneSyn For the and and p-5′ hosphoramiditeMOS- MOShemisuccinate synthesizer synthesizers -chemistrymodifieds (IOCB (IOCB Pragueal LCAA Prague,l monomers,,- CPGPrague Prague, an and,d C zech TentaGel Czechoxidation Republic) Republic) solid and sulfurization agents were used according to the supplier’s recommendations (Glen Research). usingusingsupportssulfurization 3 -3′O-O-dimethoxytritylnucleoside-5 in-dimethoxytrit a 0.5 agents µmol were scale.ylnucleoside used For the according p-5′hosphoramidite--hemisuccinate-modifiedhemisuccinate to the supplier -chemistrymodified’s recommendations al LCAAl LCAA-CPG monomers,-CPG an and dand(Glen TentaGel oxidation TentaGel Research). solid and solid Condensation0 of H-phosphinate monomers0 were performed according the synthetic protocols in supportssulfurizationCondensation in a 0.5 agents of µ µmolH-phosphinate were scale. used For the monomersaccording phosphoramidite to were the perform supplier chemistryed’s according recommendations all monomers, the synthe and (Glentic oxidation protocols Research). and in supportsTables 4 in, 5, a and 0.5 6 (molfor more scale. details For see the Supporting phosphoramidite Information chemistry). all monomers, and oxidation andsulfurizationCTableondensation sulfurizations 4, 5, and agents of 6 agents (Hfor- phosphinate weremore were useddetails used accordingmonomers see according Supporting to were to the the Information perform supplier supplier’sed’s ) according. recommendations recommendations the synthe (Glentic (Glen protocols Research). Research). in Condensation of H-phosphinate monomers were performed according the synthetic protocols in CondensationTable3.2. Hs- Phosphonate4, 5, and of 6H (-phosphinate fChemistryor more details monomers see Supporting were Information performed). according the synthetic protocols in Tables 4, 5, and 6 (for more details see Supporting Information). Table3.2.4 ,H Table-Phosphonate5, and Table Chemistry6 (for more details see Supporting Information). 3.2. HPyridine-Phosphonate-acetonitrile Chemistry (1:1 ) solutions of 0.1 M monomers (110 µL) and 0.3 M DMOCP (110 µL) Pyridine-acetonitrile (1:1) solutions of 0.1 M monomers (110 µL) and 0.3 M DMOCP (110 µL) 3.2.were H -usedPhosphonate for each Chemistry coupling step (10 min). The oxidation of the oligonucleotide chain was achieved 3.2.were H-Phosphonate Pyridineused for- acetonitrileeach Chemistry coupling (1:1 step) solutions (10 min). of The0.1 Moxidation monomers of the (110 oligonucleotide µL) and 0.3 M chain DMOCP was achieved(110 µL) usingPyridine conditions-acetonitrile in Table (21:1. In) solutionscase of methyl of 0.1 esters,M monomers the support (110 µLwas) anddried 0.3 in M vacuo, DMOCP treated (110 withµL) wereusingPyridine-acetonitrile used conditions for each in couplingTable (1:1) 2. In solutionsstep case (10 of min). ofmethyl 0.1 The M esters,oxidation monomers the ofsupport (110 the µoligonucleotideL) was and dried 0.3 M in DMOCP vacuo,chain was treated (110 achievedµ L)with were werefreshly used prepared for each thiophenol coupling -stepEt3N (10-DMF min). mixture The oxidation (23:32:45; of v the/v) foroligonucleotide 4 h, rinsed with chain dry was acetonitrile, achieved usedusingfreshly for conditions each prepared coupling inthiophenol Table step 2 (10. -InEt min). 3caseN-DMF of The methyl mixture oxidation esters, (23:32:45; of the the support oligonucleotidev/v) for was4 h, driedrinsed chain in with vacuo, was dryachieved treatedacetonitrile, with using usingdried conditionsunder vacuum, in Table and treated2. In case with of gaseousmethyl esters,NH3 (0.7 the MPa) support at rt wasovernight. dried inIn vacuo,case of 2treated-cyanoethyl with conditionsfreshlydried under prepared in Tablevacuum, 2thiophenol. In and case treated of-Et methyl3N with-DMF gaseous esters, mixture the NH (23:32:45; support3 (0.7 MPa) v was/v )at for driedrt overnight.4 h, in rinsed vacuo, In with case treated dry of 2 acetonitrile,-cyanoethyl with freshly esters was thiophenol treatment3 omitted. The deprotected and released oligonucleotide was eluted freshlydriedesters underwas prepared thiophenol vacuum, thiophenol and treatment treated-Et N omitted.with-DMF gaseous mixture The deprotectedNH (23:32:45;3 (0.7 MPa) vand/v )at forreleased rt overnight.4 h, rinsed oligonucleoti In with case dry ofde 2acetonitrile,- cyanoethylwas eluted preparedfrom CPG thiophenol-Et with 0.1 M-TEAB3N-DMF in acetonitrile/water mixture (23:32:45; (1 mLv/v); 1:1, for v 4/v h,) and rinsed analyzed with dryon a acetonitrile,DNAPac PA100 dried driedestersfrom CPGunder was withthiophenol vacuum, 0.1 M and- TEABtreatment treated in acetonitrile/water omitted.with gaseous The deprotectedNH (13 (0.7mL ;MPa) 1:1, and v at/v )releasedrt and overnight. analyzed oligonucleoti In oncase a DNAPacofde 2- cyanoethylwas PA100eluted undercolumn vacuum, (Thermo and Fisher treated Scientific with gaseous, Waltham NH, 3MA,(0.7 USA, MPa) 4 at × rt25 overnight.0 mm) at a flow In case rate of of 2-cyanoethyl 1 mL/min using esters estersfromcolumn CPGwas (Thermo thiophenolwith 0.1 Fisher M- treatmentTEAB Scientific in acetonitrile/water omitted., Waltham The, MA, deprotected (1USA, mL ;4 1:1,× 25and v0/ v mm)released) and at analyzed a flowoligonucleoti rate on ofa DNAPac1 demL was/min elutedPA100 using wasdifferent thiophenol gradients. treatment The purification omitted. The of oligonucleotide deprotected and was released performed oligonucleotide on a DNAPac wasPA100 eluted column from fromcolumndifferent CPG ( Thermogradients. with 0.1 Fisher M The-TEAB Scientificpurification in acetonitrile/water, Waltham of oligonucleotide, MA, (1USA, mL ;was4 1:1, × 25 performedv0/v mm)) and at analyzed aon flow a DNAPac rate on ofa DNAPac 1 PA100mL/min column PA100 using CPG(Thermo with 0.1 Fisher M-TEAB Scientific in acetonitrile), 9 × 250 mm/water at a flow (1 mL; rate 1:1, of v3 /mLv) and/min analyzed,). Pure olig ononucleotide a DNAPac was PA100 desalted column columndifferent(Thermo (Thermo gradients.Fisher Scientific Fisher The Scientificpurification), 9 × 250, mmWaltham of at oligonucleotide a flow, MA, rate USA, of 3 4 wasmL × 25/min performed0 mm),). Pure at a onolig flow aonucleotide DNAPac rate of 1 PA100mL was/min desalted column using (Thermoon a Luna Fisher C18 Scientific, column ( Waltham,Phenomenex, MA, Torrance USA, 4 , CA,250 mm)USA, at5 aμm; flow 10rate × 50 of mm;) 1 mL at/min a flow using rate di ffoferent 5 different(onThermo a Luna gradients.Fisher C18 Scientificcolumn The purification()Phenomenex,, 9 × 250 mm of at oligonucleotideTorrance a flow rate, CA, of 3 USAwas mL /min,performed 5 μm;,). Pure 10 ×on olig 50 a onucleotideDNAPacmm;) at a PA100 flow was ratedesaltedcolumn of 5 mL/min using a linear gradient of acetonitrile ×(0→50%, 20 min) in 0.1 M aq. triethylammonium gradients.(onmLThermo a/min Luna TheFisher using C18 purification Scientific acolumn linear ( gradient)Phenomenex,, of9 × oligonucleotide250 mm of acetonitrile at Torrance a flow was rate ,(0 CA, →performedof50%, 3 USA mL /min 2,0 5 min)μm; on,). Pure a 10 inDNAPac 0.1×olig 50 M onucleotidemm;) aq. PA100 triethylammoniumat a column flow was desaltedrate (Thermo of 5 Fisheronhydrogencarbonate a Scientific),Luna C18 9column250 buffer mm(Phenomenex, (pH at a 8); flow effluent rate Torrance of containing 3 mL, /CA,min,). oligonucleotideUSA Pure, 5 oligonucleotideμm; 10 was× 50 evaporatedmm;) was at desalteda flow to dryness rate on aof Lunaon 5 mLhydrogencarbonate/min using a × linear buffer gradient (pH 8); ofeffluent acetonitrile containing (0→50%, oligonucleotide 20 min) in was0.1 Mevaporated aq. triethylammonium to dryness on C18mLa CentriVap column/min using (Phenomenex, apparatus a linear (Labconco gradient Torrance, of, Kansas acetonitrile CA, USA,City, M 5(0µO→m;, USA50%, 10 ) 2and050 min) mm;)co-evaporated in at 0.1 a flow M aq. with rate triethylammonium MeOH of 5 mL (3/ min× 1 mL using). hydrogencarbonatea CentriVap apparatus buffer (Labconco (pH 8);, effluentKansas Citycontaining, MO, USA oligonucleotide)× and co-evaporated was evaporated with MeOH to dryness (3 × 1 mL on). a linearhydrogencarbonate gradient of acetonitrile buffer (pH (08); effluent50%, 20 containing min) in 0.1 oligonucleotide M aq. triethylammonium was evaporated hydrogencarbonate to dryness on a CentriVap apparatus (Labconco→ , Kansas City, MO, USA) and co-evaporated with MeOH (3 × 1 mL). buaff CentriVaper (pH 8); eapparatusffluent containing (Labconco oligonucleotide , Kansas City, M wasO, USA evaporated) and co- toevaporated drynesson with a CentriVap MeOH (3 × apparatus 1 mL). (Labconco, Kansas City, MO, USA) and co-evaporated with MeOH (3 1 mL). ×

Molecules 2019, 24, 1872 9 of 12

Table 4. Synthetic Protocol A (For more details see Supporting Information).

Procedure Reagents Volume/Time H-Phosphonate/H-Phosphinate Condensation Step Flow through the column Deblocking 3% DCA in DCM 6 mL/2 min 0.1M monomer in ACN/Py (1:1) Condensation 120 µL/10min 0.3M DMOCP in ACN/Py (95:5) Oxidation Ox-A, Ox-B, Ox-D 220 µL/30 min

Methyl-ester cleavage * PhSH/Et3N/DMF(1:1.4:2) 300 µL/4 h Cleavage from solid support Gaseous ammonia at 0.7 MPa Gas chamber/16 h * Applicable only in case of Ox-A and Ox-B mixtures containing methanol.

Table 5. Synthetic Protocol B (For more details see Supporting Information).

Procedure Reagents Volume/Time H-Phosphonate/H-Phosphinate Condensation Step Deblocking 3% DCA in DCM Flow through the column 6 mL/2 min DCA washout 5% pyridine in methanol 0.1M monomer in ACN/Py (1:1) Condensation 120 µL/10 min 0.3M DMOCP in ACN/Py (95:5) Oxidation or Sulfurization Ox-C or Ox-E 220 µL/30 min Ac O/Py/THF (1:1:1:17) Capping 2 2 150 µL/3 min MeIm/THF (1:9) × Phosphoramidite Condensation Step Deblocking 3% DCA in DCM Flow through the column 6 mL/2 min DCA washout 5% pyridine in methanol 0.1M monomer in ACN Condensation 120 µL/5 min 0.25M ETT in ACN Oxidation or Sulfurization CSO, tBuOOH or DDTT 220 µL/3.5 min End of Synthetic Cycle S-cyanoethyl-ester cleavage * 1M DBU in ACN 220 µL/3 min Cleavage from solid support Ammonia gas at 0.7 MPa Gas chamber/16 h * Applicable only in case of CSS sulfurization mixtures. Molecules 2019, 24, 1872 10 of 12

Table 6. Synthetic Protocol C (For more details see Supporting Information).

Procedure Reagents Volume/Time H-Phosphonate/H-Phosphinate Condensation Steps Flow through the column Deblocking 3% DCA in DCM 6 mL/2 min 0.1 M monomer in ACN/Py (1:1) Condensation 120 µL/10 min 0.3 M DMOCP in ACN/Py (95:5) Phosphoramidite Condensation Step Deblocking 3% DCA in DCM Flow through the column 6 mL/2 min DCA washout 5% pyridine in methanol 0.1 M monomer in ACN Condensation 120 µL/5 min 0.25 M ETT in ACN Oxidation CSO 220 µL/3.5 min End of Synthetic Cycle Amidation of P–H bond Ox-F 220 µL/180 min Cleavage from solid support Ammonia gas at 0.7 MPa Gas chamber/16 h

4. Conclusions In this study, we present the use of protected nucleoside-O-methyl-(H)-phosphinates as monomers for oligonucleotide synthesis using H-phosphonate chemistry. The formed O-methyl-(H)-phosphinate internucleotide linkages could be oxidized, sulfurized, or amidated similarly to the H-phosphonate bonds. We developed appropriate oxidation mixtures and three synthetic protocols allowing the combination of H-phosphonate and phosphoramidite chemistries to incorporate individual modifications either into the entire chain or at specific positions of the modified oligonucleotide. A series of DNA oligonucleotides modified with a combination of the phosphonate, phosphonothioate, phosphonamidate, phosphorothioate, phosphoramidate, and phosphodiester internucleotide linkages have been successfully synthesized to verify the robustness of the presented synthetic protocols. To our best knowledge, this is the first report of the synthesis of oligonucleotides bearing phosphonamidate and phosphonothioate internucleotide linkages. The developed protocols allow the synthesis of variety of phosphonate oligonucleotides that might be used as tools in biochemistry and biology, and in research and development of oligonucleotide-based drugs.

Supplementary Materials: The following are available online. The supplementary materials containing all experimental details. Author Contributions: Conceptualization, O.K., O.P. and I.R.; Methodology, O.K., O.P., R.L., P.N. and I.R.; Investigation O.K., P.N., and R.L.; Analysis, O.K. and P.N.; Writing and Editing, O.K., O.P. and I.R. All authors contributed to the experiments and approved the final manuscript. Funding: This research was funded by the Czech Science Foundation [17-12703S] to O.K., R.L., O.P. and I.R. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Šípová, H.; Špringer, T.; Rejman, D.; Šimák, O.; Petrová, M.; Novák, P.; Rosenbergová, Š.; Páv, O.; Liboska, R.;

Barvík, I.; et al. 50-O-Methylphosphonate nucleic acids—new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes. Nucleic Acids Res. 2014, 42, 5378–5389. [CrossRef] 2. Kostov, O.; Páv, O.; Budˇešínský, M.; Liboska, R.; Šimák, O.; Petrová, M.; Novák, P.; Rosenberg, I. 4-Toluenesulfonyloxymethyl-(H)-phosphinate: A reagent for the introduction of O- and S-Methyl-(H)- phosphinate Moieties. Org. Lett. 2016, 18, 2704–2707. Molecules 2019, 24, 1872 11 of 12

3. Kostov, O.; Páv, O.; Rosenberg, I. Nucleoside-O-methyl-(H)-phosphinates: Novel monomers for the synthesis of methylphosphonate oligonucleotides using H-phosphonate chemistry. Curr. Protoc. Nucleic Acid Chem. 2017, 70, 4.76.1–4.76.22. 4. Garegg, P.J.; Lindh, I.; Regberg, T.; Stawinski, J.; Strömberg, R.; Henrichson, C. Nucleoside H-phosphonates. III. Chemical synthesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach. Tetrahedron Lett. 1986, 27, 4051–4054. [CrossRef] 5. Froehler, B.C.; Ng, P.G.; Matteucci, M.D. Synthesis of DNA via deoxynudeoside H-phosphonate intermediates. Nucleic Acids Res. 1986, 14, 5399–5407. [CrossRef] 6. Liu, C.; Cozens, C.; Jaziri, F.; Rozenski, J.; Maréchal, A.; Dumbre, S.; Pezo, V.; Marlière, P.; Pinheiro, V.B.; Groaz, E.; et al. Phosphonomethyl oligonucleotides as backbone-modified artificial genetic polymers. J. Am. Chem. Soc. 2018, 140, 6690–6699. [CrossRef] 7. Garegg, P.J.; Regberg, T.; Stawinski, J.; Stromberg, R. Nucleoside phosphonates: Part 7. Studies on the oxidation of nucleoside phosphonate esters. J. Chem. Soc. Perkin Trans. 1 1987, 1269–1273. [CrossRef] 8. Cullis, P.M.; Lee, M. The mechanism of iodine-water oxidation of H-phosphonate diesters. J. Chem. Soc. Chem. Commun. 1992, 1207–1208. [CrossRef] 9. Wallin, R.; Kalek, M.; Bartoszewicz, A.; Thelin, M.; Stawinski, J. On the sulfurization of H-phosphonate diesters and phosphite triesters using elemental sulfur. PhosphorusSulfurSilicon Relat. Elem. 2009, 184, 908–916. [CrossRef] 10. Mohe, N.U.; Padiya, K.J.; Salunkhe, M.M. An efficient oxidizing reagent for the synthesis of mixed backbone oligonucleotides via the H-phosphonate approach. Bioorg. Med. Chem. 2003, 11, 1419–1431. [CrossRef] 11. Nilsson, J.; Stawinski, J. Oxidative coupling of H-phosphonate and H-phosphonothioate diesters. Iodine as a reagent and a catalyst. Collect. Symp. Ser. 2002, 5, 87–92. 12. Nilsson, J.; Stawinski, J. Controlling stereochemistry during oxidative coupling. Preparation of Rp or Sp from one P-chiral precursor. Chem. Commun. 2004, 2566–2567. [CrossRef] 13. Stawinski, J.; Stromberg, R. Di- and oligonucleotide synthesis using H-phosphonate chemistry. Methods Mol. Biol. (CliftonN.J.) 2005, 288, 81–100. [CrossRef] 14. Maier, M.A.; Guzaev, A.P.; Manoharan, M. Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages. Org. Lett. 2000, 2, 1819–1822. [CrossRef] 15. Bartoszewicz, A.; Kalek, M.; Stawinski, J. The Case for the intermediacy of monomeric metaphosphate analogues during oxidation of H-phosphonothioate, H-phosphonodithioate, and H-phosphonoselenoate monoesters: Mechanistic and synthetic studies. J. Org. Chem. 2008, 73, 5029–5038. [CrossRef] 16. Le Corre, S.S.; Berchel, M.; Couthon-Gourvès, H.; Haelters, J.-P.; Jaffrès, P.-A. Atherton–Todd reaction: Mechanism, scope and applications. Beilstein J. Org. Chem. 2014, 10, 1166–1196. [CrossRef] 17. Letsinger, R.L.; Singman, C.N.; Histand, G.; Salunkhe, M. Cationic oligonucleotides. J. Am. Chem. Soc. 1988, 110, 4470–4471. [CrossRef] 18. Jung, P.M.; Histand, G.; Letsinger, R.L. Hybridization of alternating cationic/anionic oligonucleotides to RNA segments. Nucleosides Nucleotides 1994, 13, 1597–1605. [CrossRef] 19. Paul, S.; Roy, S.; Monfregola, L.; Shang, S.; Shoemaker, R.; Caruthers, M.H. Oxidative substitution of boranephosphonate diesters as a route to post-synthetically modified DNA. J. Am. Chem. Soc. 2015, 137, 3253–3264. [CrossRef] 20. Brill, W.K.D. Thioalkylation of nucleoside-H-phosphonates and its application to solid phase synthesis of oligonucleotides. Tetrahedron Lett. 1995, 36, 703–706. [CrossRef] 21. Reese, C.B.; Yan, H. Solution phase synthesis of ISIS 2922 (Vitravene) by the modified H-phosphonate approach. J. Chem. Soc. Perkin Trans. 1 2002, 2619–2633. [CrossRef] 22. Alefelder, S.; Patel, B.K.; Eckstein, F. Incorporation of terminal phosphorothioates into oligonucleotides. Nucleic Acids Res. 1998, 26, 4983–4988. [CrossRef] 23. Andrus, A.; Efcavitch, J.W.; McBride, L.J.; Giusti, B. Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis. Tetrahedron Lett. 1988, 29, 861–864. [CrossRef] 24. Ugi, I.; Jacob, P.; Landgraf, B.; Ruppf, C.; Lemmen, P.; Verfürth, U. Phosphite oxidation and the preparation of five-membered cyclic phosphorylating reagents via the phosphites. Nucleosides Nucleotides 1988, 7, 605–608. [CrossRef] Molecules 2019, 24, 1872 12 of 12

25. Reynolds, M.A.; Hogrefe, R.I.; Jaeger, J.A.; Schwartz, D.A.; Riley, T.A.; Marvin, W.B.; Daily, W.J.; Vaghefi, M.M.; Beck, T.A.; Knowles, S.K.; et al. Synthesis and thermodynamics of oligonucleotides containing chirally pure R P methylphosphonate linkages. Nucleic Acids Res. 1996, 24, 4584–4591. [CrossRef] 26. Dellinger, D.J.; Sheehan, D.M.; Christensen, N.K.; Lindberg, J.G.; Caruthers, M.H. Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides. J. Am. Chem. Soc. 2003, 125, 940–950. [CrossRef] 27. Glen Research Unicap Phosphoramidite, An Alternative to Acetic Anhydride Capping. Available online: http://www.glenresearch.com/GlenReports/GR17-13.html (accessed on 9 October 2018). 28. Vlaho, D.; Damha, M.J. Synthesis of chimeric oligonucleotides having modified internucleotide linkages via an automated H-phosphonate/phosphoramidite approach. Curr. Protoc. Nucleic Acid Chem. 2018, 73, e53. [CrossRef]

Sample Availability: Not available.

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