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Retro-1-Oligonucleotide Conjugates. Synthesis and Biological Evaluation Jordi Agramunt 1 , Enrique Pedroso 1 , Silvia M. Kreda 2 , Rudolph L. Juliano 3, * and Anna Grandas 1, * 1

2 3

*

Departament de Química Inorgànica i Orgànica (Secció de Química Orgànica) and IBUB, Facultat de Química, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain; [email protected] (J.A.); [email protected] (E.P.) UNC Cystic Fibrosis Center, School of Medicine, University of North Carolina, Chapel Hill, NC 27516, USA; [email protected] UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA Correspondence: [email protected] (R.L.J.); [email protected] (A.G.); Tel.: +34-934021263 (A.G.)

Academic Editor: Roger Strömberg Received: 19 December 2018; Accepted: 1 February 2019; Published: 6 February 2019

 

Abstract: Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1–oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system. Keywords: Retro-1; oligonucleotide conjugates; antisense; splice switching

1. Introduction Forty years after the therapeutic potential of synthetic oligonucleotides was first perceived [1], delivery is the one problem that remains not well solved [2,3]. Oligonucleotides can easily be modified to favor hybridization and ameliorate their bioavailability [4,5], but there is no gold standard methodology for effective and complete internalization. Most forms of oligonucleotides are thought to be taken up by endocytosis and accumulate in intracellular endomembrane compartments [6,7]. Only a tiny fraction of the accumulated oligonucleotide spontaneously leaks from the endosomes, although this is sometimes enough to provide a pharmacological effect. There have been many efforts to increase both the cellular uptake and intracellular release of oligonucleotides including various targeting ligands and endomembrane destabilizing polymers or nanoparticles [2,8]. A few years ago, Juliano and cols. described that the compound named Retro-1 enhanced the pharmaceutical potency of antisense and siRNA oligonucleotides [9]. Retro-1 is a member of a group of compounds able to reduce the action of bacterial toxins [10] by interfering with their intracellular trafficking. Subsequently, high throughput screening has allowed identification of additional molecules capable of enhancing the effectiveness of synthetic oligonucleotides [11–13]. Some of the hits developed in these studies have been modified to assess their effect on oligonucleotide delivery [14,15]. However, to our knowledge the impact of covalently linking one of these molecules to the oligonucleotide chain has not been evaluated. Retro-1, the first compound identified to ameliorate both antisense and siRNA oligonucleotides effect, was chosen for this evaluation. In this manuscript we wish to describe the preparation of three Molecules 2019, 24, 579; doi:10.3390/molecules24030579

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[14,15]. However, to our knowledge the impact of covalently linking one of these molecules to the oligonucleotide chain has not been evaluated. Retro-1, the first compound identified to ameliorate both antisense and siRNA oligonucleotides effect, was chosen Molecules 2019, 24, 579 for this evaluation. In this manuscript we wish to describe the preparation of three 2 of 17 Retro-1 derivatives, their conjugation reactions to a splice switching oligonucleotide, and the results of the biological evaluation. These studies indicate that the efficacy of covalently linking the two Retro-1 derivatives, their reactions to a splice oligonucleotide, moieties is inferior to thatconjugation achieved by administering the switching two separate molecules. and the results of the biological evaluation. These studies indicate that the efficacy of covalently linking the two moieties is Results inferior to that achieved by administering the two separate molecules. 2. and Discussion 2. Results and Discussion 2.1. Modification of Retro-1 for Conjugation

One alternative to prepare oligonucleotide conjugates, that is, to covalently link oligonucleotides 2.1. Modification of Retro-1 for Conjugation to other molecules, involves the use of a phosphoramidite derivative that can be incorporated into One alternative to prepare oligonucleotide conjugates, that is, to covalently link oligonucleotides the chain using standard oligonucleotide elongation methodologies. Another possibility is making to other molecules, involves the use of a phosphoramidite derivative that can be incorporated into the use of a click reaction [16] that chemoselectively links the two components of the conjugate. chain using standard oligonucleotide elongation methodologies. Another possibility is making use of a Irrespective of the synthesis approach, Retro-1 had to be derivatized so as to incorporate an additional click reaction [16] that chemoselectively links the two components of the conjugate. Irrespective of the functional group. synthesis approach, Retro-1 had to be derivatized so as to incorporate an additional functional group. The structure of Retro-1 (6), which is shown on Scheme 1, has many points in common with that The structure of Retro-1 (6), which is shown on Scheme 1, has many points in common with that of benzodiazepine psychoactive drugs. It is also composed of two fused rings, where two atoms of of benzodiazepine psychoactive drugs. It is also composed of two fused rings, where two atoms of the benzene ring and carbons 6-7 of the diazepine function as bridgehead, and the 1,4-diazepine ring the benzene ring and carbons 6–7 of the diazepine function as bridgehead, and the 1,4-diazepine ring contains a carbonyl group at the 2 position and an aromatic ring appending from carbon 5. The main contains a carbonyl group at the 2 position and an aromatic ring appending from carbon 5. The main difference is that atoms 4 and 5 of the diazepine are not linked by a double bond but by a single one. difference is that atoms 4 and 5 of the diazepine are not linked by a double bond but by a single one. As a result, carbon 5 is a stereocenter. As a result, carbon 5 is a stereocenter. The synthesis of racemic Retro-1 has been described [17], as well as the separation of the two The synthesis of racemic Retro-1 has been described [17], as well as the separation of the two enantiomers using a chiral HPLC column (conformers, which are observed at the NMR spectra, could enantiomers using a chiral HPLC column (conformers, which are observed at the NMR spectra, could not be separated). Both enantiomers were shown to be biologically active, and the difference between not be separated). Both enantiomers were shown to be biologically active, and the difference between their EC50 values was small (the S isomer was a bit more active than the R one, but both were within their EC50 values was small (the S isomer was a bit more active than the R one, but both were within the same 1-10 μM range as the racemic mixture). Therefore, we have worked with the racemic the same 1–10 µM range as the racemic mixture). Therefore, we have worked with the racemic mixture. mixture. The only information available on the biological activity of Retro-1 analogs [14] indicates that The only information available on the biological activity of Retro-1 analogs [14] indicates that compounds with no substituent at the 4 position (see Scheme 1) favor oligonucleotide activity, whilst compounds with no substituent at the 4 position (see Scheme 1) favor oligonucleotide activity, whilst N-alkylation seems best suited for reducing bacterial toxins effect. Since Retro-1, in which N-4 is N-alkylation seems best suited for reducing bacterial toxins effect. Since Retro-1, in which N-4 is acylated, exhibits both effects, we decided to prepare two N-4 acylated analogs (Scheme 2) and an acylated, exhibits both effects, we decided to prepare two N-4 acylated analogs (Scheme 2) and an additional one modified at the 3 position (Scheme 3). additional one modified at the 3 position (Scheme 3). As shown in Scheme 1, the diazepine ring is made from three building blocks: the As shown in Scheme 1, the diazepine ring is made from three building blocks: the aminobenzophenone, the acylating reagent and ammonia. Acylation of the aromatic amine links aminobenzophenone, the acylating reagent and ammonia. Acylation of the aromatic amine links the the two main components and imine formation closes the cycle. two main components and imine formation closes the cycle.

Scheme 1. Synthesis scheme described [17] for the preparation of Retro-1, 6. DCM = dichloromethane; NBS = N-bromosuccinimide; TEA = triethylamine. The wavy bond indicates that the stereochemistry is not defined (in other words, the compound is a mixture of isomers).

Based on this scheme, which we could perfectly reproduce, changing the reagents of the two acylation reactions appeared to be a simple option to adapt Retro-1 for conjugation. Replacement of

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Scheme 1. Synthesis scheme described [17] for the preparation of Retro-1, 6. DCM = dichloromethane; NBS = N-bromosuccinimide; TEA = triethylamine. The wavy bond indicates that the stereochemistry is not defined (in other words, the compound is a mixture of isomers).

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Based on this scheme, which we could perfectly reproduce, changing the reagents of the two acylation reactions appeared be a simple option to adapt Retro-1 for conjugation. Replacement of bromoacetyl bromide with an to activated amino acid would modify position 3 of the diazepine ring, and bromoacetyl bromide with an activated amino acid would modify position 3 of the diazepine ring, use of a suitably protected trifunctional amino acid would incorporate an additional group suitable for and usederivatization. of a suitably Alternatively, protected trifunctional amino acid would incorporate an additional group further substitution of propionyl chloride with another acylation reagent suitable for further derivatization. Alternatively, substitution of propionyl chloride with another would allow position 4 to be modified. acylation reagentthat would positionanalogs 4 to be modified. We decided the allow three Retro-1 would be differently derivatized for conjugation. One We decided that the three Retro-1 analogs would differently derivatized One would include a phosphitylatable hydroxyl group, and be could be attached to the 5’for endconjugation. of a resin-linked would include a phosphitylatable hydroxyl group, and could be attached to the 5' end of a resinchain. The other two would contain functional groups suitable for click conjugations in solution. For linked chain.we The other two and would contain which functional click conjugations in this purpose chose a thiol a 1,3-diene, were groups intendedsuitable to reactfor with a maleimide moiety. solution. For this purpose we chose a thiol and a 1,3-diene, which were intended to react with a Maleimido-oligonucleotides can be easily assembled on a solid support making use of the appropriate maleimide moiety. Maleimido-oligonucleotides can be easily assembled on a solid support making maleimide protection [18]. use of the appropriate maleimide protection [18]. 2.1.1. Retro-1 Derivatives Modified at the 4 Position 2.1.1. Retro-1 derivatives modified at the 4 position Two of the three analogs were synthesized from the amine precursor of Retro-1 5 (see Scheme 1). Two of the three analogs were with synthesized the amine of Retro-1 5 (seebe Scheme Replacement of propionyl chloride acryloylfrom chloride gave 7 precursor (Scheme 2a), which could easily 1). Replacement of propionyl chloride with acryloyl chloride gave 7 (Scheme 2, a), which could be modified by means of Michael-type reactions. easilyReaction modifiedbetween by means of Michael-type reactions.afforded 8, from which phosphoramidite 9 was 7 and 4-hydroxypiperidine Reaction between 7 and 4-hydroxypiperidine afforded 8, from phosphoramidite 9 was prepared by reaction with a chlorophosphine and a base (Scheme 2b). which The other aza-Michael reaction prepared by reaction with a chlorophosphine and a base (Scheme 2, b). The other aza-Michael was carried out with S-trityl cysteamine, which furnished 10, and removal of the trityl group under reaction was carried out 11 with S-trityl2c). cysteamine, whichthe furnished 10,thiol and removal ofto thedisulfide trityl group acidic conditions gave (Scheme To minimize extent of oxidation the under acidic conditions gave 11 (Scheme 2, c). To minimize the extent of thiol oxidation to disulfide thiol-containing Retro-1 analog was kept protected, and thiol deprotection was carried out not long the thiol-containing Retro-1 analog was kept protected, and thiol deprotection was carried out not before the conjugation reaction. long before the conjugation reaction.

Scheme 2.

Synthesis of the Retro-1 analogs modified at position 4 of the benzodiazepine

Scheme 2. Synthesis of the Retro-1 analogs modified at position 4 of the benzodiazepine ring. The first step (a) afforded the common precursor, which was subsequently modified ring. The first step (a) afforded thederivative common precursor, which was subsequently modified to obtain either phosphoramidite 9 (b) or the thiol-modified compound 11 (c). to obtain either phosphoramidite derivative 9 (b) or the thiol-modified compound (c). CNE = 2-cyanoethyl; DCM = dichloromethane; DIPEA = N,N-diisopropylethylamine; rt =11room CNE = 2-cyanoethyl; DCM = dichloromethane; DIPEA = N,N-diisopropylethylamine; = temperature; TEA = triethylamine; TFA = trifluoroacetic acid; TIS = triisopropylsilane; Trt = trityl. rt The wavy bond indicates a not defined stereochemistry.

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room temperature; TEA = triethylamine; TFA = trifluoroacetic acid; TIS = triisopropylsilane;4 of 17 Molecules 2019, 24, 579 Trt = trityl. The wavy bond indicates a not defined stereochemistry. 2.1.2. Modification of the 3 Position of Retro-1 startingmaterial material the preparation of the thirdderivative Retro-1 derivative the first The starting for for the preparation of the third Retro-1 was the firstwas intermediate intermediate inofthe synthesis Retro-1, 2 (see Scheme of theprotected amine with in the synthesis Retro-1, 2 (seeofScheme 1). Acylation of the 1). amineAcylation with a suitably lysinea suitably protected lysine derivative was foundAttempts not to betostraightforward. Attempts to activate derivative was found not to be straightforward. activate the carboxyl group with either carboxyl group either a combination of a carbodiimide and 1-hydroxybenzotriazole, athe combination of with a carbodiimide and 1-hydroxybenzotriazole, 1-[(1-cyano-2-ethoxy-21-[(1-cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylaminomorpholinomethylene)] methanoxoethylideneaminooxy)-dimethylaminomorpholinomethylene)] methanaminium aminium hexafluorophosphate (COMU), or mesitylenesulfonyl 3-nitro-1,2,4-triazole (MSNT) in hexafluorophosphate (COMU), or mesitylenesulfonyl 3-nitro-1,2,4-triazole (MSNT) in the presence theN-methylimidazole presence of N-methylimidazole were unsuccessful. the mixed carbonic carboxylic carbonic of were unsuccessful. Gratifyingly,Gratifyingly, the mixed carboxylic anhydride anhydride methodology did work 3). (Scheme 3). Carboxyl group activation with isobutyl chloroformate methodology did work (Scheme Carboxyl group activation with isobutyl chloroformate in the in the presence of N-methylmorpholine afforded 12, overnight and overnight treatment ofwith 12 with M ammonia presence of N-methylmorpholine afforded 12, and treatment of 12 7 M7 ammonia in in methanol quantitatively removed theFmoc Fmocgroup groupand andpromoted promotedcyclization cyclizationto to 13. 13. These These two two steps methanol quantitatively removed the were also carried out without isolating isolating 12, 12, and and 13 13 was was obtained obtained in in aa similar similar yield. yield.

Synthesis of diene-derivatized 16. = acetonitrile; ACN = acetonitrile; Scheme 3.3. Synthesis of diene-derivatized Retro-1Retro-1 analog analog 16. ACN Boc = tertBoc = tert-butoxycarbonyl; DCM = dichloromethane; diene-COOH = (4E)-4,6-heptadienoic butoxycarbonyl; DCM = dichloromethane; diene-COOH = (4E)-4,6-heptadienoic acid; DIPC = N,N'acid; DIPC = N,N’-diisopropylcarbodiimide; Fmoc NMM = 9-fluorenylmethoxycarbonyl; diisopropylcarbodiimide; Fmoc = 9-fluorenylmethoxycarbonyl; = N-methylmorpholine; on = NMM = N-methylmorpholine; on =The overnight; rt = indicates room temperature. Thestereochemistry. wavy bond indicates a not overnight; rt = room temperature. wavy bond a not defined defined stereochemistry.

Diene-derivatized 16 was finally obtained after the following series of steps: i) Reduction of the Diene-derivatized 16 was finally obtained after the following series of steps: (i) Reduction of the imine of compound 13 with NaBH3CN as previously reported, which gave 14; ii) Removal of the Boc imine of compound 13 with NaBH3 CN as previously reported, which gave 14; (ii) Removal of the group with an acidic treatment, which yielded 15; iii) Carbodiimide-promoted acylation of the Boc group with an acidic treatment, which yielded 15; (iii) Carbodiimide-promoted acylation of the primary amine with (4E)-4,6-heptadienoic acid. We were initially concerned with the possible primary amine with (4E)-4,6-heptadienoic acid. We were initially concerned with the possible acylation acylation of the secondary amine (N-4 of the diazepine ring), but we found that reaction took place of the secondary amine (N-4 of the diazepine ring), but we found that reaction took place exclusively exclusively on the lysine ε-amine, furnishing 16. In fact, all attempts carried out to acylate N-4 failed. on the lysine ε-amine, furnishing 16. In fact, all attempts carried out to acylate N-4 failed. Therefore, Therefore, derivative 16 differed from the other Retro-1 analogs in the presence of a substituent derivative 16 differed from the other Retro-1 analogs in the presence of a substituent appending from appending from carbon 3 and no acyl group on nitrogen 4. carbon 3 and no acyl group on nitrogen 4. 2.2. Preparation of the Retro-1 Conjugates The three Retro-1 derivatives (9, 11, 16) were linked to oligonucleotide r5’GTTATTCTTTAGAATGGTGC3’ (all 2’-O-methyl and phosphorothioate, T = ribothymidine). This

introduced into the firefly luciferase gene used for the splice switching experiments [19]. Compounds 9, 11 and 16 were also linked to a control oligonucleotide with a scrambled sequence, namely r5'TGTGTACTGATGTAGTTATC3' (all 2'-O-methyl and phosphorothioate; T = ribothymidine). After elongation of the two oligonucleotide chains using standard methodology and BTT as Molecules 2019, 24, =579 5 of 17 activator (BTT 5-benzylthio-1H-tetrazole), removal of the DMT group (DMT = 4,4'-dimethoxytrityl) on the 5' end furnished oligonucleotide-resins 17. This was followed by coupling of phosphoramidite 9 (2 × 10 min coupling, activation with BTT) and sulfurization (Beaucage reagent). The resulting sequence is complementary to that of a mutated intron from thalassemic hemoglobin that is introduced Retro-1-oligonucleotide-resins (18) were treated with conc. aq. ammonia (3 h, rt) to yield conjugates into the firefly luciferase gene used for the splice switching experiments [19]. Compounds 9, 19a and 19b (Scheme 4). 11 and 16 were also linked to a control oligonucleotide with a scrambled sequence, namely For the preparation of the other conjugates (Scheme 5), the 2,5-dimethylfuran-protected r5’TGTGTACTGATGTAGTTATC3’ (all 2’-O-methyl and phosphorothioate; T = ribothymidine). maleimide phosphoramidite was coupled to either oligonucleotide-resin 17 (2 × 10 min coupling, After elongation of the two oligonucleotide chains using standard methodology and BTT as activation with BTT), and the resulting phosphite triester sulfurized (Beaucage reagent). Treatment activator (BTT = 5-benzylthio-1H-tetrazole), removal of the DMT group (DMT = 4,4’-dimethoxytrityl) of oligonucleotide-reins 20 with conc. aq. ammonia afforded [protected maleimido]-oligonucleotides on the 5’ end furnished oligonucleotide-resins 17. This was followed by coupling of phosphoramidite 21, which were purified. Conjugates 22 and 23 were obtained by simultaneously carrying out 9 (2 × 10 min coupling, activation with BTT) and sulfurization (Beaucage reagent). The resulting maleimide deprotection (retro Diels-Alder reaction) and reaction with either the thiol-containing Retro-1-oligonucleotide-resins (18) were treated with conc. aq. ammonia (3 h, rt) to yield conjugates derivative 11 (Michael-type reaction) or the diene-derivatized compound 16 (Diels-Alder 19a and 19b (Scheme 4). cycloaddition) in a microwave (MW) oven.

Scheme 4. Solid-phase assembly of conjugates 19. B = G (oligonucleotide a)/T (oligonucleotide b); Scheme 4. Solid-phase assembly of conjugates 19. B = G (oligonucleotide a) / T (oligonucleotide b); BTT = 5-benzylthio-1H-tetrazole; CNE = 2-cyanoethyl; PS = phosphorothioate. The wavy bond indicates BTT = 5-benzylthio-1H-tetrazole; CNE = 2-cyanoethyl; PS = phosphorothioate. The wavy bond a not defined stereochemistry. indicates a not defined stereochemistry.

For the preparation of the other conjugates (Scheme 5), the 2,5-dimethylfuran-protected maleimide phosphoramidite was coupled to either oligonucleotide-resin 17 (2 × 10 min coupling, activation with BTT), and the resulting phosphite triester sulfurized (Beaucage reagent). Treatment of oligonucleotide-reins 20 with conc. aq. ammonia afforded [protected maleimido]-oligonucleotides 21, which were purified. Conjugates 22 and 23 were obtained by simultaneously carrying out maleimide deprotection (retro Diels-Alder reaction) and reaction with either the thiol-containing derivative 11 (Michael-type reaction) or the diene-derivatized compound 16 (Diels-Alder cycloaddition) in a microwave (MW) oven. All conjugates were purified by reversed phase HPLC and characterized by MALDI-TOF MS.

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Scheme 5. 5.Preparation of conjugates conjugates2222and and G (oligonucleotide a)/T (oligonucleotide Scheme Preparation of 23.23. B =B G= (oligonucleotide a) / T (oligonucleotide b); BTT b); BTT = 5-benzylthio-1H-tetrazole; CNE = 2-cyanoethyl; MW = microwave; PS = phosphorothioate. The = 5-benzylthio-1H-tetrazole; CNE = 2-cyanoethyl; MW = microwave; PS = phosphorothioate. The wavy bond indicates wavy bond indicatesa anot notdefined definedstereochemistry. stereochemistry.

2.3. Splice-Switching All conjugatesAssays were purified by reversed phase HPLC and characterized by MALDI-TOF MS. Oligonucleotides conjugated to Retro-1 derivatives were examined for splice correction activity. 2.3. Splice-Switching Assays The experiments utilized the HeLa Luc 705 cell line [19], as described in Materials and Methods. Oligonucleotides having the known r5’GTTATTCTTTAGAATGGTGC3’, Oligonucleotides conjugated to splice Retro-1correcting derivativessequence were examined for splice correction activity. The experiments utilized[19], the HeLa Luc 705 cell line [19], as described inform Materials andcompared Methods. to previously termed SSO623 were used in conjugated or unconjugated and were ', Oligonucleotides havingwith the known splicesequence correctingr5’TGTGTACTGATGTAGTTATC3’. sequence r5'GTTATTCTTTAGAATGGTGC3' control oligonucleotides the inactive In Figure 1 previously termed SSO623 [19], were used in conjugated or unconjugated form and were compared oligonucleotide 19a, which is the immediate conjugate of Retro-1, was compared to its control to control19b. oligonucleotides with inactive sequence r5'TGTGTACTGATGTAGTTATC3'. In Figure by conjugate Furthermore, wethe examined unmodified SSO623, as well as SSO623 followed 1 oligonucleotide 19a, molecule which is UNC7938 the immediate conjugate of Retro-1, was to its control treatment with the small as a positive control. As seen in compared the figure, oligonucleotide conjugate 19b. Furthermore, we examined unmodified SSO623, as well as SSO623 followed 19a produced a dose-dependent luciferase induction while the control oligonucleotide 19b didbynot. treatment with the small molecule UNC7938 as a positive control. As seen in the figure, However, SSO623 itself also gave rise to a progressive induction of luciferase that was somewhat oligonucleotide 19a produced a dose-dependent luciferase induction while the control greater than that produced by 19a. The dual use of SSO623 and UNC7938 provided the largest degree oligonucleotide 19b did not. However, SSO623 itself also gave rise to a progressive induction of of splice correction and luciferase induction as expected. This set of experiments demonstrated that luciferase that was somewhat greater than that produced by 19a. The dual use of SSO623 and coupling of the Retro moiety did not prevent the splice correcting activity of the active oligonucleotide. UNC7938 provided the largest degree of splice correction and luciferase induction as expected. This However, conjugate 19a did not provide an advantage, in terms of potency or efficacy, over SSO623 set of experiments demonstrated that coupling of the Retro moiety did not prevent the splice itself. We also examined two additional conjugates 22a and 23a. However, neither of these conjugates correcting activity of the active oligonucleotide. However, conjugate 19a did not provide an displayed an advantage SSO623 in the HeLa 705 induction advantage, in terms ofover potency or efficacy, over Luc SSO623 itself. We assay. also examined two additional conjugates 22a and 23a. However, neither of these conjugates displayed an advantage over SSO623 in the HeLa Luc 705 induction assay.

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Figure 1. HeLa705 cells in complete growth medium were incubated for 16 h with various concentrations of conjugated or unconjugated oligonucleotide. Thereafter the cells were recovered and assayed for luciferase activity. In one case cells were post-treated with 10 µM UNC7938 for 4 h after Figure 1. HeLa705 cells in complete growth medium were incubated for 16 h with various exposure to SSO623 and then assayed. RLU = relative luminescence units; n = 3. Means and standard concentrations of conjugated or unconjugated oligonucleotide. Thereafter the cells were recovered errors shown. SSO623: r5’GTTATTCTTTAGAATGGTGC3’, all 2’-O-Me and phosphorothioate (PS) and assayed for luciferase activity. In one case cells were post-treated with 10 μM UNC7938 for 4 h (T = ribothymidine). after exposure to SSO623 and then assayed. RLU = relative luminescence units; n = 3. Means and ', all 2'-O-Me standard errors shown.of the SSO623: r5'GTTATTCTTTAGAATGGTGC3' In summary, conjugation Retro moiety to a splice switching oligonucleotide did notand provide phosphorothioate (PS) (T = ribothymidine). a major enhancement of splice correction activity in the widely used HeLa Luc705 reporter system.

At this point it is unclear why the conjugates displayed slightly lower activity than the unmodified In summary, conjugation of the Retro moiety to a splice switching oligonucleotide did not oligonucleotide. One could hypothesize that the presence of the bulky Retro group might affect provide a major enhancement of splice correction activity in the widely used HeLa Luc705 reporter either cell uptake of the oligo or might affect the interaction of the oligo with the splicing machinery. system. At this point it is unclear why the conjugates displayed slightly lower activity than the However, detailed investigation of these possibilities, especially the splicing aspect, would involve a unmodified oligonucleotide. One could hypothesize that the presence of the bulky Retro group might very substantial amount of new biological investigation and is beyond the scope of this work. affect either cell uptake of the oligo or might affect the interaction of the oligo with the splicing machinery. However, detailed investigation of these possibilities, especially the splicing aspect, 3. Experimental Section would involve a very substantial amount of new biological investigation and is beyond the scope of 3.1. Materials and Methods this work. 3.1.1. Materials for Solution Organic Synthesis 3. Experimental Section (E)-Hepta-4,6-dienoic acid was prepared as described by Baillie et al. [20], and S-trityl cysteamine 3.1. Materialsbyand Methodset al. [21]. Fmoc-L-Lys(Boc)-OH was from Novabiochem (Zaragoza, Spain); all as reported Naumiec of the other chemicals were either from Sigma-Aldrich (Zaragoza, Spain) or Across Organics (Zaragoza, 3.1.1. Materials for Solution Organic Synthesis Spain) (7 M ammonia in methanol), and were used without further purification. Water was obtained from(E)-Hepta-4,6-dienoic a MilliQ system (Zaragoza, acid Spain). was prepared as described by Baillie et al. [20], and S-trityl cysteamine as reported by Naumiec et al. [21]. Fmoc-L-Lys(Boc)-OH was from Novabiochem (Merck, 3.1.2. Analysis andother Characterization Techniques Small Organic Molecules (2–16)Spain) or Across Spain); all of the chemicals were either for from Sigma-Aldrich (Millipore, Organics M ammonia in 60 methanol), and were(Zaragoza, used without further purification. TLC (Millipore, was carriedSpain) out on(7silica gel plates F254 from Merck Spain). IR spectra were Water was MilliQ system (Millipore, recorded inobtained a Nicoletfrom 6700 aFT-IR spectrometer (ThermoSpain). Scientific, Zaragoza, Spain). 1 H and 13 C NMR spectra were recorded on either Varian Mercury 400 MHz or Brucker 400 MHz spectrometers (reference: 3.1.2. andsolvent Characterization forspectra Small Organic Molecules (2–16) TMS Analysis or residual signals). Techniques HR-ESI mass were obtained using an LC/MSD-TOF Instrument (Agilent Technologies, Zaragoza, Spain). HPLC/MS analyses were recorded an Alliance TLC was carried out on silica gel plates 60 F254 from Merck (Millipore, Spain). IRin spectra were Waters 2690 separation module with a Waters micromass ZQ4000 MS detector (Waters, Zaragoza, 1 13 recorded in a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, Millipore, Spain). H and C NMR Spain). Aqueous solutions in either Labconco or Christ spectra were recorded on were eitherlyophilized Varian Mercury 400 MHz or(Vertex, BruckerZaragoza, 400 MHzSpain) spectrometers freeze dryers (Inycom, Zaragoza, Spain). (reference: TMS or residual solvent signals). HR-ESI mass spectra were obtained using an LC/MSD-

TOF instrument (Agilent Technologies, Millipore, Spain). HPLC/MS analyses were recorded in an 3.1.3. Materials for Oligonucleotide Assembly Alliance Waters 2690 separation module with a Waters micromass ZQ4000 MS detector (Waters, 0 -OMe Ac , iPrPac , Nucleoside phosphoramidites AinPaceither , C G(Vertex, and 5-MeU), Millipore, Spain). Aqueous solutions were(2lyophilized Labconco Millipore, Spain) 0 0 Ac 5 -O-DMT-2 -OMe-C glass beads, and oligonucleotide synthesis reagents were or Christ freeze dryers -succinyl-derivatized (Inycom, Millipore, Spain). from Link Technologies (Manchester, UK). [Protected maleimido]-phosphoramidite (see structure in Scheme 5) was synthesized as previously described [18].

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3.1.4. Oligonucleotide Assembly Oligonucleotide chains were elongated in a 3400 ABI automatic synthesizer at the 1 µmol scale, using standard phosphite triester methodology. The activator used at the coupling step was 5-benzylthio-1H-tetrazole (0.3 M solution in anh. acetonitrile, 10 min coupling time), and the Beaucage reagent was used for sulfurization (0.05 M in anh. acetonitrile; 2 × 4 min). All phosphoramidites were dissolved in anh. acetonitrile (0.1 M solutions), except that of 20 -OMe-5-MeU (which was dissolved in anh. DCM). Final deprotection conditions: conc. aq. ammonia, 3 h, room temperature. After this treatment the resin was filtered and washed with water (3×), the filtrates pooled and concentrated in a SpeedVac apparatus (Inycom, Zaragoza, Spain) (removal of ammonia), and the resulting crude lyophilized. 3.1.5. RP-HPLC Analysis and Purification of Oligonucleotides and Conjugates Reversed-phase HPLC analysis and purification was performed using a Shimadzu system (Izasa, Zaragoza, Spain). Linear gradients were always used. Analysis conditions for oligonucleotides and conjugates were Jupiter C18 column (10 µm, 300 Å, 250 × 4.6 mm) from Phenomenex (Zaragoza, Spain), solvent A: 0.1 M aq. triethylammonium acetate, solvent B: acetonitrile, flow: 1 mL/min, detection wavelength: 254 nm. Semipreparative purification conditions: Jupiter C18 column (10 µm, 300 Å, 250 × 10.0 mm) from Phenomenex, solvent A: 0.1 M aq. triethylammonium acetate, solvent B: acetonitrile, flow: 3 mL/min, detection wavelength: 254 nm. 3.1.6. MALDI-TOF MS of Oligonucleotides and Conjugates MALDI-TOF mass spectra were recorded on a 4800 Plus instrument (AB Sciex, Zaragoza, Spain), not using the reflector mode unless otherwise indicated. Typical analysis conditions: 1:1 (v/v) 2,4,6-trihydroxyacetophenone/ammonium citrate (THAP/CA), negative mode. 3.2. Synthesis of Small Molecules (Compounds 8–16) Compounds 1–6 were Synthesized as Previously Described [17]. 7-Bromo-1,3,4,5-tetrahydro-4-(acryloyl)-5-phenyl-2H-1,4-benzodiazepin-2-one (7). To a suspension of 5 (150 mg, 0.47 mmol) in DCM (5 mL) acryloyl chloride (50 µL, 0.62 mmol) was added, and the mixture stirred at room temperature for 3 h. Afterwards, additional DCM (30 mL) was added, and the mixture was transferred to a separatory funnel and washed with H2 O (3 × 20 mL). The organic layer was dried over anh. MgSO4 , filtered, and the solvent removed under low pressure. The resulting crude was purified by silica gel flash column chromatography eluting with DCM/EtOAc mixtures from 100:0 to 75:25. The title compound (7) was obtained as a white foam (130 mg, 74%). TLC (DCM/EtOAc 80:20): Rf = 0.50; IR (ATR, solid): 3221, 2920, 2359, 2340, 1682, 1647, 1515, 1416, 665 cm−1 ; 1 H NMR (CDCl3 , 400 MHz): δ 8.88 and 8.85 (s, 1H), 7.45 (s, 1H), 7.46 and 7.40 (d, J = 8.5 Hz, 1H), 7.32–7.24 (m, 3H), 7.05 (s, 1H), 7.04 and 6.16 (s, 1H), 6.93 and 6.89 (d, J = 8.5 Hz, 1H), 6.61 and 6.54 (dd, J = 10.4, 16.4 Hz, 1H), 6.45 and 6.41 (s, 1H), 5.84–5.78 (m, 1H), 4.34–4.02 (m, 2H) ppm; diastereomer 1, 13 C NMR (CDCl3 , 101 MHz): δ 170.39, 166.04, 138.44, 134.30, 132.09, 130.61, 129.02, 127.93, 126.67, 122.91, 117.43, 59.46, 48.72 ppm; diastereomer 2, 13 C NMR (CDCl3 , 101 MHz): δ 170.39, 166.63, 137.52, 135.52, 134.71, 133.15, 132.73, 130.86, 130.00, 129.06, 128.39, 128.21, 127.08, 126.79, 123.67, 117.55, 63.49, 46.30 ppm; ESI-HRMS (positive mode): m/z 371.0391/373.0368 (81 Br) [M + H]+ , M calcd for C18 H16 BrN2 O2 371.0390. 7-Bromo-1,3,4,5-tetrahydro-4-[1-oxo-3-(4-hydroxypiperidin-1-yl)propyl]-5-phenyl-2H-1,4-benzodiazep-in-2-one (8). 7 (100 mg, 0.27 mmol) and 4-hydroxypiperidine (273.1 mg, 2.70 mmol) were dissolved in DCM (5 mL) and reacted overnight at room temperature. Afterwards, the solvent was removed under low pressure, the resulting crude dissolved in EtOAc (40 mL) and washed with H2 O (3 × 20 mL). The

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organic phase was dried over anh. MgSO4 , filtered, and the solvent removed under vacuum. The title compound (8) was obtained as a white solid (125 mg, 98%). TLC (DCM:EtOAc 50:50): Rf = 0.20; IR (ATR, solid): 3214, 3119, 2983, 2353, 2334, 1865, 1650, 1558, 1553, 1508, 1239, 783, 666 cm−1 ; 1 H NMR (CDCl3 , 400 MHz): δ 8.70 and 8.68 (s, 1H), 7.46–7.38 (m, 2H), 7.34–7.27 (m, 3H), 7.06–7.00 (m, 2H), 6.95 and 6.20 (s, 1H), 6.92–6.87 (m, 1H), 4.45–3.98 (m, 2H), 3.65 (p, J = 4.5 Hz, 1H), 2.80–2.54 (m, 6H), 2.20–2.12 (m, 2H), 1.87–1.79 (m, 4H), 1.57–1.47 (m, 1H) ppm; diastereomer 1, 13 C NMR (CDCl3 , 101 MHz): δ 171.64, 170.40, 138.33, 134.86, 134.25, 132.05, 130.34, 129.14, 128.96, 127.74, 122.87, 117.39, 67.49, 63.07, 59.29, 53.75, 51.19, 34.18, 31.60 ppm; diastereomer 2, 13 C NMR (CDCl , 101 MHz): δ 171.96, 170.94, 137.60, 135.50, 133.17, 132.60, 130.37, 128.44, 128.10, 3 127.05, 123.45, 117.19, 67.44, 60.42, 53.82, 51.30, 46.10, 34.19, 31.30 ppm; ESI-HRMS (positive mode): m/z 472.1222/474.1205 (81 Br) [M + H]+ , M calcd for C23 H27 BrN3 O3 472.1230. 7-Bromo-1,3,4,5-tetrahydro-4-[1-oxo-3-(4-hydroxypiperidin-1-yl)propyl]-5-phenyl-2H-1,4-benz-odiazepin-2-one, 2-cyanoethyl N,N-diisopropylphosphoramidite (9). 8 (453.5 mg, 0.96 mmol) was dissolved in anh. DCM (15 mL). Subsequently, anh. N,N-diisopropylethylamine (310 µL, 2.41 mmol) was added and the mixture was stirred in an ice bath for 5 min under an argon atmosphere. Afterwards, a solution of chloro(2-cyanoethoxy)diisopropylaminophosphine (250 mg, 1.06 mmol) in anh. DCM (1 mL) was added, and the mixture was reacted in an ice bath for 30 min. Then, it was allowed to warm up and left stirring for 6 h at room temperature until complete phosphitylation as shown by TLC. The solvent was removed under low pressure; the crude was dissolved in EtOAc (50 mL) and washed with aq. NaHCO3(sat.) (3 × 30 mL). The organic phase was dried over anh. MgSO4 , filtered, and the solvent removed under low pressure. The resulting crude was further purified by silica gel flash column chromatography eluting with DCM/EtOAc/NEt3 mixtures from 98:0:2 to 48:50:2. The title compound (9) was obtained as a white foam (272.4 mg, 42%). TLC (DCM/EtOAc/NEt3 48:50:2): Rf = 0.50; 1 H NMR (CDCl3 , 400 MHz): δ 7.90 and 7.86 (s, 1H), 7.48–7.40 (m, 2H), 7.33–7.27 (m, 3H), 7.05–7.01 (m, 2H), 6.95 and 6.22 (s, 1H), 6.88–6.80 (m, 1H), 4.44–4.00 (m, 2H), 3.90–3.71 (m, 3H), 3.65–3.45 (m, 2H), 2.80–2.56 (m, 8H), 2.38–2.23 (m, 2H), 1.93–1.79 (m, 2H), 1.76–1.60 (m, 2H), 1.27 and 1.17 (dd, J = 6.8, 5.5 Hz, 12H) ppm; 31 P NMR (CDCl3 , 162 MHz): δ 145.85 ppm; ESI-HRMS (positive mode): m/z 672.2298/674.2281 (81 Br) [M + H]+ , M calcd for C32 H44 BrN5 O4 P 672.2309. 7-Bromo-5-phenyl-4-{3-[(2-(tritylthio)ethyl)amino]propanoyl}-1,3,4,5-tetrahydro-2H-benzo[e][1,4]di-azepin2-one (10). 7 (370 mg, 0.99 mmol) and S-tritylcysteamine (1.50 g, 4.49 mmol) were dissolved in DCM (5 mL) and reacted overnight at room temperature. Afterwards, the solvent was removed under low pressure, and the resulting crude was purified by silica gel flash column chromatography eluting with hexanes/EtOAc/MeOH mixtures from 30:70:0 to 0:100:3. The title compound (10) was obtained as a white solid (441 mg, 64%). TLC (EtOAc/MeOH 97:3): Rf = 0.35; IR (ATR, solid): 3211, 2926, 1729, 1666, 1664, 1482, 1448, 1368, 1242, 1216, 1188, 1058, 821, 745, 694 cm−1 ; 1 H NMR (CDCl3 , 400 MHz): δ 7.44–7.40 (m, 9H), 7.30–7.18 (m, 14H), 7.03–6.99 (m, 1H), 6.90 and 6.11 (s, 1H), 4.42–3.96 (m, 2H), 2.80–2.66 (m, 2H), 2.60–2.45 (m, 3H), 2.41–2.31 (m, 3H), 1.87 (br s, 1H) ppm; diastereomer 1, 13 C NMR (CDCl3 , 101 MHz): δ 171.49, 169.75, 145.01, 138.34, 134.80, 134.45, 133.43, 132.28, 130.38, 129.15, 128.00, 127.85, 127.13, 126.80, 122.89, 117.61, 66.71, 63.16, 48.73, 44.67, 41.11, 33.67, 31.86 ppm; diastereomer 2, 13 C NMR (CDCl3 , 101 MHz): δ 171.86, 170.35, 144.90, 137.43, 135.47, 133.43, 132.83, 130.41, 129.33, 128.62, 128.30, 128.04, 127.13, 126.80, 123.40, 117.61, 66.80, 63.16, 48.40, 44.89, 41.11, 33.31, 31.81 ppm; ESI-HRMS (positive mode): m/z 690.1776/692.1765 (81 Br) [M + H]+ , M calcd for C39 H37 BrN3 O2 S 690.1784. 7-Bromo-4-{3-[(2-mercaptoethyl)amino]propanoyl}-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diaze-pin2-one (11). 10 (20 mg, 0.03 mmol) was dissolved in a mixture of TFA/TIS (95:5) and reacted at room temperature for 90 min. Afterwards, the solvent was removed under a N2 stream and the resulting crude dissolved in a mixture MeOH/H2 O (1:1, 2 mL) and filtered through a hydrophilic PTFE

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temperature for 90 min. Afterwards, the solvent was removed under a N2 stream and the resulting crude dissolved in a mixture MeOH/H2O (1:1, 2 mL) and filtered through a hydrophilic PTFE Molecules 2019, 24, 579 10 of 17 (polytetrafluoroethylene) syringe filter (0.22 µm), lyophilized and used without further purification (quantitative thiol deprotection as assessed by HPLC, Figure 2). (polytetrafluoroethylene) syringe filter (0.22 µm), lyophilized and used without further purification HPLC-MS: Analysis conditions: 0 → 50 % B in 30 min, tR = 19.0 min. ESI-MS (positive mode) of (quantitative thiol deprotection 81 as assessed by HPLC, Figure 2).

the main peak: m/z 448.2 / 450.2 ( Br), M calcd for C20H23BrN3O2S 448.07.

.

Figure 2. Analytical HPLC trace of crude 11 (detection wavelength: 250 nm).

HPLC-MS: Analysis conditions: 0→ 50 % in 3011 min, tR = 19.0 min. ESI-MS (positive Figure 2. Analytical HPLC trace of B crude (detection wavelength: 250 nm). mode) of the main peak: m/z 448.2/450.2 (81 Br), M calcd for C20 H23 BrN3 O2 S 448.07.

α ε -L-lysinyl)-2-amino-5-bromobenzophenone (12). N-Methylmorpholine (200 µL, 1.81 mmol) -Fmoc-N N-(NN-(N -Fmoc-N -L-lysinyl)-2-amino-5-bromobenzophenone (12). N-Methylmorpholine (200 µL, 1.81 and isobutylchloroformate (94 µL, were added to a solution of Fmocmmol) and isobutylchloroformate (940.73 µL,mmol) 0.73 mmol) were added to a solution ofL-Lys(Boc)-OH Fmoc-L-Lys(Boc)(340.5 mg, 0.73 mmol) in anh. DCM (5 mL), and the mixture cooled in an ice bath. After 15 min, OH (340.5 mg, 0.73 mmol) in anh. DCM (5 mL), and the mixture cooled in an ice bath. After 15 min, a solution of 2 (200.0 mg, 0.73 mmol) in anh. DCM (1 mL) was added and the mixture was stirred for a solution of 2 (200.0 mg, 0.73 mmol) in anh. DCM (1 mL) was added and the mixture was stirred for 1 h at 5 ◦ C and overnight at room temperature. Afterwards, further DCM was added (15 mL) and the o 1 h at 5 C and at room temperature. further DCM (15 mL) and the mixture wasovernight washed with aq. HCl 10% (2 × 10Afterwards, mL). The organic phase waswas driedadded over anh. MgSO 4, 4, mixture was washed with aq. HCl 10% (2 × 10 mL). The organic phase was dried over anh. MgSO filtered and the solvent evaporated under low vacuum. The resulting crude was purified by silica filtered and the solvent evaporated under vacuum. The resulting was purified by silica gel flash column chromatography elutinglow with DCM/MeOH mixturescrude from 100:0 to 97:3. The title gel flashcompound column chromatography withfoam DCM/MeOH mixtures from 100:0 to 97:3. The title (12) was obtained aseluting a pale white (301 mg, 57%). compound (12) was obtained as a pale white foam (301 mg, 57%). TLC (DCM/MeOH 95:5): R = 0.40; IR (ATR, solid): 2980, 2905, 2355, 1730, 1658, 1599, 1571, 1497, 1425, α

ε

f

1 H NMR (CDCl , 400 MHz): δ 1391, 1254, 95:5): 1248, 1173, 1158,IR 1071, 1043, 757, 744, 694, 533 2355, cm−1 ;1730, 3 1571, 1497, 1425, TLC1437, (DCM/MeOH Rf = 0.40; (ATR, solid): 2980, 2905, 1658, 1599, (d, J1254, = 8.8 Hz, 1H), 7.85–7.52 9H), 1043, 7.46–7.23 7H),694, 5.95533 (br. cm s, 1H), 4.78–4.63 1H),3,4.55–4.34 -1; 1H NMR (m, (CDCl 400 MHz): δ 1437,8.57 1391, 1248, 1173, 1158,(m, 1071, 757,(m, 744, (m, 2H), 4.32–4.20 (m, 2H), 3.13 (t, J = 8.0 Hz, 2H), 2.02 (m, 2H), 1.93–1.78 (m, 2H), 1.77–1.50 (m,1H), 2H),4.55– 8.57 (d, J = 8.8 Hz, 1H), 7.85–7.52 (m, 9H), 7.46 – 7.23 (m, 7H), 5.95 (br. s, 1H), 4.78–4.63 (m, 13 C NMR (CDCl , 101 MHz): δ 197.84, 171.21, 156.47, 156.31, 143.69, 141.23, 138.78, 1.45 (s, 9H) ppm; 4.34 (m, 2H), 4.32–4.20 (m, 2H), 3.133 (t, J = 8.0 Hz, 2H), 2.02 (m, 2H), 1.93–1.78 (m, 2H), 1.77–1.50 (m, 137.60, 136.75, 135.51, 132.91, 129.94, 128.46, 127.73, 127.66, 127.09, 127.05, 125.47, 125.14, 120.01, 119.89, 2H), 1.45 (s, 9H) ppm; 13C NMR (CDCl3, 101 MHz): δ 197.84, 171.21, 156.47, 156.31, 143.69, 141.23, 115.13, 79.21, 77.48, 77.16, 76.84, 67.44, 56.46, 47.20, 39.77, 31.73, 29.81, 28.47, 22.42 ppm. ESI-HRMS 138.78, 137.60, 136.75, 135.51, 132.91, 129.94, 128.46, 127.73, 127.66, 127.09, 127.05, 125.47, 125.14, (positive mode): m/z 726.2173/728.2161 (81 Br) [M + H]+ ; M calcd for C39 H40 BrN3 O6 726.2173.

120.01, 119.89, 115.13, 79.21, 77.48, 77.16, 76.84, 67.44, 56.46, 47.20, 39.77, 31.73, 29.81, 28.47, 22.42 ppm. Tert-Butyl (S)-[4-(7-bromo-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)butyl]carbamate (13). 12 ESI-HRMS (positive mode): m/z 726.2173 / 728.2161 (81Br) [M + H]+; M calcd for C39H40BrN3O 6 726.2173. (150 mg, 0.21 mmol) was dissolved in a 7 M ammonia solution (in MeOH, 3 mL) and reacted at room

Tert-Butyl (S)-[4-(7-bromo-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)butyl]carbamate temperature overnight. Afterwards, the reaction mixture was taken to dryness and the crude purified (13). 12 (150 mg, 0.21 mmol) was chromatography dissolved in a 7eluting M ammonia solution (in MeOH, mixture. 3 mL) and by silica gel flash column with a 70:30 hexanes/EtOAc Thereacted title at roomcompound temperature overnight. the solid reaction mixture taken to dryness the crude (13) was obtainedAfterwards, as a pale yellow (104 mg, 99%).was For characterization seeand below. purified by silica gel flash column chromatography eluting with a 70:30 hexanes/EtOAc mixture. The One-pot synthesis of compound 13 from 2. A solution of Fmoc-L-Lys(Boc)-OH (1.87 g, 4.00 mmol) in anh. title DCM compound (13) was obtained pale yellow solid (104 mg, (1.37 99%).mL, For9.09 characterization see below. (15 mL) was cooled in anas icea bath. N-Methylmorpholine mmol) and isobutyl chloroformate mL, 3.64 added.of After 10 Lmin, a solution of 2 (1.00 g, 3.64 mmol) One-pot synthesis of(0.81 compound 13mmol) from 2.were A solution Fmoc-Lys(Boc)-OH (1.87 g, 4.00 mmol) in anh. ◦ in anh. DCM (2 mL) was added, and the mixture was stirred for 30 min at 5 C and 5.5 h at room DCM (15 mL) was cooled in an ice bath. N-Methylmorpholine (1.37 mL, 9.09 mmol) and isobutyl chloroformate (0.81 mL, 3.64 mmol) were added. After 10 min, a solution of 2 (1.00 g, 3.64 mmol) in anh. DCM (2 mL) was added, and the mixture was stirred for 30 min at 5 oC and 5.5 h at room

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temperature. Afterwards, the solvent was removed under vacuum and the crude dissolved in 7 M NH3 (in MeOH, 40 mL), and the solution was left to react overnight at room temperature. Subsequently, the solvent was removed under vacuum and the resulting crude dissolved in EtOAc (100 mL) and washed with 10% aq. HCl (3 × 30 mL). The organic phase was dried over anh. MgSO4 , filtered and the solvent evaporated under vacuum. The crude material was further purified by silica gel flash column chromatography eluting with a 70:30 hexanes/EtOAc mixture. The title compound (13) was obtained as a pale yellow solid (970 mg, 55%). TLC (hexanes/EtOAc 1:1): Rf = 0.42; IR (ATR, solid): 3414, 3309, 2929, 2359, 2337, 1682, 1653, 1508, 1232, 1156, 669 cm−1 ; 1 H NMR (CDCl3 , 400 MHz): δ 9.45 (s, 1H), 7.60 (dd, J = 8.6, 2.3 Hz, 1H), 7.52–7.34 (m, 6H), 7.08 (d, J = 8.6 Hz, 1H), 4.63 (s br, 1H), 3.50 (dd, J = 8.2, 5.7 Hz, 1H), 3.19 (d, J = 7.6 Hz, 2H), 2.32–2.14 (m, 2H), 1.69–1.56 (m, 2H), 1.44 (s, 11H) ppm; 13 C NMR (CDCl3 , 101 MHz): δ 172.13, 168.09, 156.03, 138.64, 137.65, 134.57, 133.30, 130.51, 129.67, 129.10, 128.34, 123.04, 116.00, 79.01, 63.22, 40.47, 30.66, 30.00, 28.44, 23.32 ppm; ESI-HRMS (positive mode): m/z 486.1386/488.1370 (81 Br) [M + H]+ , M calcd for C24 H29 BrN3 O3 486.1387. 7-Bromo-1,3,4,5-tetrahydro-4-[4-(N-Boc-amino)butyl]-5-phenyl-2H-1,4-benzodiazepin-2-one (14). To a solution of 13 (800 mg, 1.65 mmol) and NaBH3 CN (155.4 mg, 2.48 mmol) in MeOH (10 mL), AcOH (500 µL, 8.24 mmol) was added, and the mixture was stirred at room temperature for 2.5 h until complete reduction of the imine as assessed by TLC. Afterwards, the solvent was removed under low pressure, the crude was dissolved in EtOAc (50 mL) and washed with aq. NaHCO3(sat) (2 × 20 mL). The organic layer was dried over anh. MgSO4 , filtered and the solvent removed under low pressure. The title compound (14) was obtained as a pale yellow solid (799 mg, 99%). TLC (hexanes/EtOAc 1:1): Rf = 0.52; IR (ATR, solid): 3290, 2967, 2929, 2866, 1663, 1479, 1365, 1245, 1159, 817, 700 cm−1 ; 1 H NMR (CDCl3 , 400 MHz): δ 7.99 and 7.67 (s, 1H), 7.45–7.27 (m, 5H), 7.13 and 6.76 (d, J = 2.3 Hz, 1H), 6.89 and 6.80 (d, 8.4 Hz, 1H), 5.34 and 5.24 (s, 1H), 4.56 (br s, 1H), 3.57 and 3.31 (t, J = 7.2 Hz, 1H), 3.14–3.05 (m, 2H), 1.94–1.44 (m, 6H), 1.42 (s, 9H) ppm; diastereomer 1, 13 C NMR (CDCl3 , 101 MHz): δ 175.02, 156.00, 142.23, 139.97, 136.56, 132.23, 128.64, 128.45, 127.19, 122.69, 117.53, 79.06, 63.98, 59.94, 40.29, 31.65, 29.93, 28.41, 23.50 ppm; diastereomer 2, 13 C NMR (CDCl3 , 101 MHz): δ 174.27, 156.00, 142.33, 139.97, 135.88, 131.35, 128.57, 128.20, 127.54, 123.32, 118.88, 79.06, 58.97, 56.14, 40.29 31.65, 30.04, 28.42, 23.21 ppm; ESI-HRMS (positive mode): m/z 488.1559/490.1555 (81 Br) [M + H]+ , M calcd for C24 H31 BrN3 O3 488.1543. 7-Bromo-1,3,4,5-tetrahydro-4-(4-aminobutyl)-5-phenyl-2H-1,4-benzodiazepin-2-one dihydrochloride (15). In a 10 mL round-bottom flask, 14 (100 mg, 0.21 mmol) was dissolved in 4 M HCl (in dioxane, 5 mL) and the reaction mixture was left stirring for 2.5 h at room temperature. Afterwards the solvent was removed under low pressure to afford the title product (15) as a pale yellow solid (94 mg, 99%). TLC (DCM/MeOH 9:1): Rf = 0.20; IR (ATR, solid): 3474, 3199, 2911, 2711, 2524, 1704, 1590, 1568, 1479, 1448, 1375, 1315, 1270, 1245, 1131, 998, 916, 827, 697 cm−1 ; 1 H NMR (CD3 OD, 400 MHz): δ 7.75 and 7.73 (s, 1H), 7.69–7.55 (m, 4H), 7.44–7.38 (m, 1H), 7.34–7.29 (m, 2H), 7.19 (d, J = 8.5 Hz) and 7.09 (d, J = 9.2 Hz, 1H), 6.02 and 5.71 (s, 1H), 4.12 (dd, J = 9.0, 4.6 Hz) and 3.86 (dd, J = 10.5, 3.1 Hz, 1H), 2.99–2.89 (m, 2H), 2.32–2.10 (m, 1H), 2.00–1.80 (m, 1H), 1.71 (h, J = 7.3 Hz, 2H), 1.58–1.34 (m, 2H) ppm; diastereomer 1, 13 C NMR (CDCl , 101 MHz): δ 166.45, 138.28, 135.43, 135.17, 133.98, 130.67, 130.20, 129.58, 130.20, 3 129.58, 128.34, 125.45, 120.24, 60.83, 57.09, 40.24, 28.13, 27.96, 23.88 ppm; diastereomer 2, 13 C NMR (CDCl3 , 101 MHz): δ 167.82, 136.80, 135.87, 134.92, 133.34, 131.13, 130.35, 129.58, 128.34, 126.16, 119.84, 63.60, 57.38, 40.24, 29.17, 28.05, 23.66 ppm; ESI-HRMS (positive mode): m/z 388.1016/390.1000 (81 Br) [M + H]+ , M calcd for C19 H23 BrN3 O 388.1019. 7-Bromo-1,3,4,5-tetrahydro-4-{4-[N-[(4E)-4,6-heptadienoyl]-amino]butyl}-5-phenyl-2H-1,4-benzodiazepin-2-one (16). N,N’-Diisopropylcarbodiimide (61 µL, 0.39 mmol) and N-methylmorpholine (2.6 µL, 0.03 mmol) were added to a solution of (4E)-4,6-heptadienoic acid (49 mg, 0.39 mmol) in HPLC-quality acetonitrile (1 mL), and the mixture was left stirring for 10 min. Afterwards, 15 (50 mg, 0.13 mmol) and an

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additional amount of N-methylmorpholine (2.6 µL, 0.03 mmol) were poured onto the mixture. Molecules 2018, 23, x of 17 Additional amounts of N-methylmorpholine (2.6 µL, 0.03 mmol) were added after 20 and 4012min, respectively, and the solution was stirred for up to 2 h at room temperature. Subsequently, acetonitrile respectively, and the up a tohydrophilic 2 h at room temperature. Subsequently, was added (4 mL), and solution the crudewas wasstirred filteredfor using PTFE syringe filter (0.22 µm), acetonitrile was added (4 mL),Proteo and the crude was filtered hydrophilic syringe filter (0.22 purified by RP-HPLC (Jupiter C18 (10 µm, 250 nm,using 250 ×a 10 mm) fromPTFE Phenomenex, solvent µm), purified by RP-HPLC (Jupiter Proteo C18 (10 μm, 250 nm, 250 × 10 mm) from Phenomenex, A: H2 O 0.1% formic acid; solvent B: ACN 0.1% formic acid, linear gradient 40 → 80% B, 3 mL/min, solvent A: H2O 0.1%254 formic solvent B: ACN 0.1% formic acid, gradientas40 → 80 solid %B, 3 detection wavelength nm) acid; and lyophilized. The title compound (16) linear was obtained a white mL/min, detection wavelength 254 nm) and lyophilized. The title compound (16) was obtained as a (10.7 mg, 20%). white solid (10.7 mg, 20%). TLC (EtOAc/MeOH 9:1): Rf = 0.10; IR (ATR, solid): 2980, 2905, 2355, 1730, 1658, 1599, 1571, 1497, 1425, 1; 1H TLC 1391, (EtOAc/MeOH Rf =1158, 0.10; 1071, IR (ATR, solid): 2905, 2355, 1730, 1658, 1599, 1571,, 400 1497, 1425, 1437, 1254, 1248,9:1): 1173, 1043, 757, 2980, 744, 694, 533 cm− NMR (CDCl MHz) 3 −1; 1H NMR (CDCl3, 400 MHz) δ 1437, 1391, 1254, 1248, 1173, 1158, 1071, 1043, 757, 744, 694, 533 cm δ 7.65–7.53 (m, 4H), 7.42–7.28 (m, 4H), 7.16 and 7.04 (d, J = 8.5 Hz, 1H), 7.47 and 6.97 (d, J = 2.2 Hz, 7.65–7.53 (m, 4H), 7.42–7.28 (m, 4H), 7.16 and J =(s,8.51H), Hz,5.70–5.61 1H), 7.47 (m, and1H), 6.97 5.06 (d, J (dd, = 2.2 JHz, 1H), 1H), 6.35–6.21 (m, 1H), 6.13–6.02 (m, 1H), 5.80 7.04 and (d, 5.68 = 16.9, 6.35–6.21 (m, 1H), 6.13–6.02 (m, 1H), 5.80 and 5.68 (s, 1H), 5.70–5.61 (m, 1H), 5.06 (dd, J = 16.9, 1.9 Hz, 1.9 Hz, 1H), 4.92 (dt, J = 10.2, 2.2 Hz, 1H), 4.17–3.77 (m, 1H), 3.72 (dd, J = 10.1, 3.7 Hz, 1H), 3.23–3.07 13 1H), 4.92 (dt, J = 10.2, 2.2 Hz, 1H), 4.17–3.77 (m, 1H), 3.72 (dd, J = 10.1, 3.7 Hz, 1H), 3.23 – 3.07 (m, 2H), (m, 2H), 2.39–2.31 (m, 2H), 2.27–2.12 (m, 3H), 1.81–1.63 (m, 1H), 1.55–1.31 (m, 4H) ppm; C NMR 2.39–2.31 (m, 2H), 2.27–2.12 (m, 3H), 1.81–1.63 (m, 1H), 1.55–1.31 (m, 4H) ppm; 13C NMR (CDCl3, 101 (CDCl 3 , 101 MHz): δ 173.99, 173.93, 136.91, 135.65, 133.59, 132.93, 132.50, 131.86, 129.62, 129.28, 128.67, MHz):126.88, δ 173.99, 173.93, 136.91, 135.65, 132.93, 132.50, 131.86, 129.28, 128.67, 127.77, 127.77, 123.98, 118.74, 114.45, 59.34,133.59, 55.99, 38.24, 35.25, 29.37, 28.76,129.62, 28.34, 22.80 ppm; ESI-HRMS + , M 29.37, 126.88, 123.98, 114.45, 59.34, 55.99, 35.25, 22.80 ppm; ESI-HRMS (positive mode): 118.74, m/z 496.1584/498.1573 (81 Br)38.24, [M + H] calcd. 28.76, for C2628.34, H30 BrN 3 O3 496.1594. (positive mode): m/z 496.1584 / 498.1573 (81Br) [M + H]+, M calcd. for C26H30BrN3O3 496.1594. RP-HPLC analysis conditions (Figure 3): Jupiter Proteo C18 (4 µm, 254 nm, 250 × 4.6 mm) from RP-HPLC 3): Jupiter Proteo 18 (4 μm, 254 nm, 250 × 4.6 mm) from Phenomenex, 30analysis → 70% Bconditions in 30 min,(Figure 1 mL/min, tR = 14.3 min C and 18.1 min; purification conditions: Phenomenex, 30 → 70 %B in 30 min, 1 mL/min, t R = 14.3 min and 18.1 min; purification conditions: Jupiter Proteo C18 (10 µm, 254 nm, 250 × 10 mm) from Phenomenex, 40 → 80% B in 30 min, 3 mL/min Proteo C18 (10 μm, 254 nm, 250 × 10 mm) from Phenomenex, 40 → 80 %B in 30 min, 3 mL/min (tJupiter R = 7.5 min and 8.9 min). (tR = 7.5 min and 8.9 min).

Figure 3. Analytical HPLC traces of crude 16 (top), purified diastereomer 1 (bottom, left) and purified diastereomer 2 (bottom, right). Detection wavelength: 250 nm. Top trace, tR ~ 2–3 min: 15 & N,N’-diisopropylurea.

3.3. Preparation of Oligonucleotides and Conjugates r5’GTTATTCTTTAGAATGGTGC3’ (2’-OMe, PS) (purchased from Avecia, Manchester, UK). HPLC analysis conditions (Figure 4): 0 → 50% B in 30 min, tR = 19.0 min. Figure 3. Analytical HPLC traces of crude 16 (top), purified diastereomer 1 (bottom, left) and purified diastereomer 2 (bottom, right). Detection wavelength: 250 nm. Top trace, tR ~ 2–3 min: 15 & N,N'diisopropylurea.

3.3. Preparation of Oligonucleotides and Conjugates

Molecules 2018, 2018, 23, 23, x x Molecules Molecules 2018, 23, x

13 of of 17 17 13 13 of 17

Molecules 2019, 24, 579 13 of 17 r5'GTTATTCTTTAGAATGGTGC3' (2’-OMe, PS) (purchased from Avecia, Manchester, United r5'GTTATTCTTTAGAATGGTGC3' (2’-OMe, (purchased fromtRAvecia, Manchester, United Kingdom). HPLC analysis conditions (Figure 4): 0 PS) → 50% B in 30 min, = 19.0 min. R Kingdom). HPLC analysis conditions (Figure 4): 0 → 50% B in 30 min, tR = 19.0 min.

Figure 4. Analytical HPLC trace of oligonucleotide r5’GTTATTCTTTAGAATGGTGC3’. Detection Figure 4. Analytical HPLC trace of oligonucleotide r5'GTTATTCTTTAGAATGGTGC3'. Detection wavelength: 250 nm. HPLC trace of oligonucleotide r5'GTTATTCTTTAGAATGGTGC3'. Detection Figure 4. Analytical wavelength: 250 nm. wavelength: 250 nm. −

MALDI-TOF MS (negative mode, THAP/CA): m/z 7060.9 [M − H] , M calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7060.9 [M-H]−−, M calcd. for C218 H BrN O P S 7053.8. 290 69 124 19 19 MALDI-TOF MS7053.8. (negative mode, THAP/CA): m/z 7060.9 [M-H]−, M calcd. for C218 290 218Hr5’TGTGTACTGATGTAGTTATC3’ 290BrN69 69O124 124P19 19S19 19 (2’-OMe, PS). HPLC: Analysis conditions, 0 → 50% B in 30 min, C218H290BrN69O124P19S19 7053.8. r5'TGTGTACTGATGTAGTTATC3' (2’-OMe,20PS). HPLC: → 50% B in 30 tR = 19.2 min (Figure 5). Purification conditions: → 40% B inAnalysis 30 min (tconditions, = 9.0 min).00Yield (synthesis R r5'TGTGTACTGATGTAGTTATC3' (2’-OMe, PS). HPLC: Analysis conditions, → 50% B in 30 R = 19.2 min (Figure 5). Purification conditions: 20 → 40% B in 30 min (tR R = 9.0 min). Yield min, t R and purification): 43%. min, t R = 19.2 min (Figure 5). Purification conditions: 20 → 40% B in 30 min (tR = 9.0 min). Yield (synthesis and purification): 43%. (synthesis and purification): 43%.

Figure 5. Analytical HPLC HPLC trace trace of Detection Figure 5. Analytical of oligonucleotide oligonucleotide r5’TGTGTACTGATGTAGTTATC3’. r5'TGTGTACTGATGTAGTTATC3'. Detection Figure 5. Analytical HPLC trace of oligonucleotide r5'TGTGTACTGATGTAGTTATC3'. Detection wavelength: 250 nm. wavelength: 250 nm. wavelength: 250 nm.

MALDI-TOF MS 7053.8 [M [M-H] − H]−−−,, M for MALDI-TOF MS (negative (negative mode, mode,THAP/CA): THAP/CA):m/zm/z 7053.8 M calcd. calcd. for − C218 O12419P19 MALDI-TOF MS (negative mode, THAP/CA): m/z 7053.8 [M-H] , M calcd. for 218 H 290 N 69124 19 S 19 7053.8. 290 69O C218 H290 N69 124P19 S19 7053.8. Oligonucleotide-resins were automatically assembled at the 1 µmol-scale as stated C218HConjugates 290N69O124P19. 19S19 7053.8. Conjugates 19. Oligonucleotide-resins were automatically assembled at the 1 μmol-scale as stated Conjugates 19. Oligonucleotide-resins wereoligonucleotide automatically assembled atwas the completed 1completed μmol-scale as stated above (Materials and Methods section). After oligonucleotide elongation was and the 5' 5’ above (Materials and Methods section). After elongation and the above (Materials phosphoramidite and Methods section). oligonucleotide elongation was completed2and 5' end deprotected, 9 was After incorporated (0.1 M solution in anh. acetonitrile, × 10the min end deprotected, phosphoramidite 9 was incorporated (0.1 M solution in anh. acetonitrile, 2 × 10 min end deprotected, 9 was incorporated (0.1 M solution inprocedure anh. acetonitrile, × 10other min coupling) and thephosphoramidite resulting phosphite triester sulfurized using the same as in all2the coupling) and the resulting phosphite triester sulfurized using the same procedure as in all the other coupling) cycles, and thewhich resulting phosphite triester sulfurizedwas using the same as in all the other synthesis cycles, which afforded 18. Final Final deprotection was carried outprocedure under standard standard conditions synthesis afforded 18. deprotection carried out under conditions synthesis cycles, which afforded 18. Final deprotection was carried out under standard conditions (see above). (see above). (see above). Conjugate 19a. HPLC HPLC (Figure (Figure 6): 6): Analysis Analysis conditions: conditions: 00 → → 50% 50% BB in in 30 30 min, min, ttRR == 22.0 22.0 min; min; Conjugate 19a. R Conjugate 19a. HPLC 6):30Analysis 0 → (synthesis 50% B in and 30 min, tR = 22.0 36%. min; purification conditions: 0 →(Figure 40% B in min (t =conditions: 19.1 min). Yield purification): purification conditions: 0 → 40% B in 30 min (tRRR= 19.1 min). Yield (synthesis and purification): 36%. purification conditions: 0 → 40% B in 30 min (tR = 19.1 min). Yield (synthesis and purification): 36%.

Figure 6. HPLC trace of crude (left) and purified (right) conjugate 19a. Detection wavelength: 254 Figure6.6.HPLC HPLCtrace trace crude (left) and purified (right) conjugate 19a. Detection wavelength: Figure ofof crude (left) and purified (right) conjugate 19a. Detection wavelength: 254254 nm. nm. nm.

MALDI-TOF MS (negative mode, THAP/CA): m/z 7610.5 [M − H]− , M calcd. C241 H315 BrN72 O128 P20 S20 7602.8.

for

Molecules 2018, 2018, 23, 23, xx Molecules Molecules 2018, 23, 579 x 2019, 24,

14 of of 17 17 14 14 14 of of 17

MALDI-TOF MS (negative mode, THAP/CA): m/z 7610.5 [M-H]−−−, M calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7610.5 [M-H] , M calcd. for 241H315 315BrN72 72O128 128P20 20 7602.8. C241 19b.20S20 HPLC (Figure 7): Analysis conditions, 0 → 50% B in 30 min, tR = 21.7 min; C241HConjugate 315BrN72O128P20S20 7602.8. Conjugate 19b. HPLC (Figure Analysis → 50% B in 30 min, tRR = 21.7 min; purification conditions: 20 → 40% B 7): in 30 min (tR conditions, = 13.8 min). 00Yield (synthesis and purification): 20%. Conjugate 19b. HPLC (Figure 7): Analysis conditions, → 50% B in 30 min, tR = 21.7 min; purification conditions: 20 → 40% B in 30 min (tRR = 13.8 min). Yield (synthesis and purification): 20%. purification conditions: 20 → 40% B in 30 min (tR = 13.8 min). Yield (synthesis and purification): 20%.

Figure 7. HPLC trace of crude (left) and purified (right) conjugate 19b. Detection wavelength: 254 nm. Figure 7. 7. HPLC HPLC trace trace of of crude crude (left) (left) and and purified purified (right) (right) conjugate conjugate 19b. 19b. Detection Detection wavelength: wavelength: 254 254 Figure Figure 7. tHPLC of crude (left) and purified (right) conjugate 19b. Detection wavelength: 254 Left trace, min: r5’TGTGTACTGATGTAGTTATC3’. R ~ 19 trace R ~ 19 min: r5'TGTGTACTGATGTAGTTATC3'. nm. Left trace, t nm. Left trace, tR ~ 19 min: r5'TGTGTACTGATGTAGTTATC3'. nm. Left trace, tR ~ 19 min: r5'TGTGTACTGATGTAGTTATC3'.

MALDI-TOF MS (negative mode, THAP/CA): m/z 7602.1 [M − H]− , M calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7602.1 [M-H]−−−, M calcd. for C241 H BrN O P S 7602.8. , M calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7602.1 [M-H] 315 72 128 20 20 241H315 315BrN72 72O128 128P20 20S20 20 7602.8. C241 Oligonucleotide-resins were assembled as previously described. The C241HOligonucleotides 315BrN72O128P20S2021. 7602.8. Oligonucleotides 21. Oligonucleotide-resins were assembled as previously described. The [2,5[2,5-dimetylfuran-protected maleimide]-phosphoramidite (see structure in Schemedescribed. 5) was subsequently Oligonucleotides 21. Oligonucleotide-resins were assembled as previously The [2,5dimetylfuran-protected maleimide]-phosphoramidite (see structure in Scheme 5) was subsequently incorporated (0.1 M solution in anh. acetonitrile, 2 × 10 inmin coupling), which after dimetylfuran-protected maleimide]-phosphoramidite (see structure Scheme 5) was subsequently incorporated (0.1 M solution in anh. acetonitrile, 2 × 10 min coupling), which after sulfurization sulfurization (0.1 afforded 20. Treatment with ammonia (asmin described above) furnished [protected incorporated M solution in anh. acetonitrile, 2 × 10 coupling), which after sulfurization afforded 20. Treatment with ammonia (as described above) furnished [protected maleimido]maleimido]-oligonucleotides afforded 20. Treatment with21.ammonia (as described above) furnished [protected maleimido]oligonucleotides 21. Oligonucleotide oligonucleotides 21. 21a. HPLC (Figure 8): Analysis conditions: 0 → 50% B in 30 min, tR = 19.7 min; Oligonucleotide 21a. HPLC (Figure 8): Analysis conditions: 0 → 50% B in 30 min, tRR = 19.7 min; purification conditions: → 40% B in 30 (tR = 10.7 min). Yield purification): 26%. Oligonucleotide 21a.20 HPLC (Figure 8):min Analysis conditions: 0 →(synthesis 50% B inand 30 min, tR = 19.7 min; purification conditions: 20 → 40% B in 30 min (tRR = 10.7 min). Yield (synthesis and purification): 26%. purification conditions: 20 → 40% B in 30 min (tR = 10.7 min). Yield (synthesis and purification): 26%.

Figure HPLC traces of crude (left) and purified (right) oligonucleotide 21a. Detection Figure8. 8. 8. HPLC HPLC traces of crude crude (left) and purified purified (right) oligonucleotide oligonucleotide 21a. wavelength: Detection Figure traces of (left) and (right) 21a. Detection Figure 8. HPLC traces of crude (left) and purified (right) oligonucleotide 21a. Detection 254 nm. wavelength: 254 254 nm. nm. wavelength: wavelength: 254 nm.

MALDI-TOF MS (negative mode, THAP/CA): m/z 7375.1 [M − H]− , M calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7375.1 [M-H]---, M calcd. for , M calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7375.1 [M-H] C230 H N O P S 7368.8. 304 70 129 20 20 230H304 304N70 70O129 129P20 20S20 20 7368.8. C230 C230HOligonucleotide 304N70O129P20S20 21b. 7368.8. HPLC (Figure 9): Analysis conditions, 0 → 50% B in 30 min, t = 20.2 min; Oligonucleotide 21b. HPLC (Figure 9): Analysis conditions, 0 → 50% B in 30 min, tRRR = 20.2 min; Oligonucleotide 21b.20 HPLC (Figure Analysis conditions, 0→ 50% B inand 30 min, tR = 20.2 min; min). Yield (synthesis purification): 20%. purification conditions: → 40% B in 309): min (t = 11.3 purification conditions: 20→ 40% B in 30 min (tRRR = 11.3 min). Yield (synthesis and purification): 20%. purification conditions: 20→ 40% B in 30 min (tR = 11.3 min). Yield (synthesis and purification): 20%.

Figure 9. HPLC traces of crude (left) and purified (right) oligonucleotide 21b. Detection wavelength: Figure 9. 9. HPLC HPLC traces traces of of crude crude (left) (left) and and purified purified (right) (right) oligonucleotide oligonucleotide 21b. 21b. Detection Detection wavelength: wavelength: Figure Figure HPLC traces (left) and purified (right) oligonucleotide 21b. Detection wavelength: 254 nm.9.Left trace, tR ~of 19crude min: r5’TGTGTACTGATGTAGTTATC3’. R ~ 19 min: r5'TGTGTACTGATGTAGTTATC3'. 254 nm. Left trace, t 254 nm. Left trace, tR ~ 19 min: r5'TGTGTACTGATGTAGTTATC3'. 254 nm. Left trace, tR ~ 19 min: r5'TGTGTACTGATGTAGTTATC3'.

MALDI-TOF MS (negative mode, THAP/CA): m/z 7369.0 [M − H]− , M calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7369.0 [M-H]---, M calcd. for C230 H N O P S 7368.8. MALDI-TOF MS (negative mode, THAP/CA): m/z 7369.0 [M-H] , M calcd. for 304 70 129 20 20 230H304 304N70 70O129 129P20 20S20 20 7368.8. C230 To a solution of 21a (260 nmol) in MeOH/H2 O (1:1, 468 µL) a solution of C230HConjugate 304N70O129P22a. 20S20 7368.8. Conjugate 22a. To a solution of 21a (260 nmol) in MeOH/H22O (1:1, 468 µL,) a solution of 11 (520 11 (520 nmol, 22a. 52 µL, mM) inofMeOH/H was added, and 468 the µL,) resulting mixture Conjugate To 10 a solution 21a (260 nmol) in MeOH/H 2O (1:1, a solution of 11(final (520 2 O (1:1) nmol, 52 µL, 10 mM) in MeOH/H22O (1:1) was added, and the resulting mixture (final oligonucleotide nmol, 52 µL, 10 mM) in MeOH/H2O (1:1) was added, and the resulting mixture (final oligonucleotide

Molecules 2019, 24, 579 Molecules 2018, 23, x Molecules 2018, Molecules 2018, 23, x23, x

15 of 17 15 of 17 15 17 of 17 15 of

oC in a microwave concentration 140 µM) heated at 90 for 90 min. was oligonucleotide= concentration = 140 µM) heated at 90 ◦ C in oven a microwave ovenAfterwards, for 90 min. MeOH Afterwards, o concentration = 140 µM) heated at 90 Cthe a microwave oven min. Afterwards, MeOH was concentration = 140reduced µM) heated at 90 and C oin ainmicrowave oven forfor 90 90 min. Afterwards, MeOH was removed under pressure resulting crude purified by HPLC. HPLC (Figure 10): MeOH was removed under reduced pressure and the resulting crude purified by HPLC. HPLC removed under reduced pressure and the resulting crude purified by HPLC. HPLC (Figure 10): removed under reduced pressure and the resulting crude purified by HPLC. HPLC (Figure 10): Analysis10): conditions, → 50% B in030→min, 22.4 conditions: 20 →conditions: 40% B in 3020min (Figure Analysis0conditions, 50%tRB= in 30min, min,purification tR = 22.4 min, purification → Analysis conditions, 0 → 50% B in 30 min, t R = 22.4 min, purification conditions: 20 → 40% B in 30 min Analysis conditions, 0 → 50% B in and 30 min, tR = 22.4 min, purification conditions: 20 → 40% B in 30 min (t R = 14.4 Yield purification): 11%. 40% B in min). 30 min (tR =(synthesis 14.4 min). Yield (synthesis and purification): 11%. = 14.4 min). Yield (synthesis purification): 11%. (tR =(tR14.4 min). Yield (synthesis andand purification): 11%.

Figure 10. HPLC traces of crude (left) and purified (right) conjugate 22a. Detection wavelength: Figure 254 nm.10. HPLC traces of crude (left) and purified (right) conjugate 22a. Detection wavelength: 254 Figure HPLC traces of crude (left) purified (right) conjugate Detection wavelength: Figure 10. 10. HPLC traces of crude (left) andand purified (right) conjugate 22a.22a. Detection wavelength: 254254 nm. − nm. nm.MALDI-TOF MS (negative mode, THAP/CA): m/z 7720.7 [M − H] , M calcd. for

MALDI-TOF mode, THAP/CA): m/z 7720.7 [M-H]−, M calcd. for C244 H 7719.8. 318 BrN73 O130 PMS 20 S21 (negative −, − MALDI-TOF MS (negative mode,THAP/CA): THAP/CA):m/zm/z7720.7 7720.7[M-H] [M-H] calcd.forfor MALDI-TOF (negative MM calcd. C244 HConjugate 318BrN73O130 PMS 20S21 23a. To7719.8. a solutionmode, of 21a (800 nmol) in MeOH/H2 O (1:1, 18, mL) 16 was added CH244318HConjugate 318BrN OP23a. 130 P2120To S21 7719.8. C244(4000 BrN 73O73 130 20S a solution of 21a (800 nmol) in (final MeOH/H 2O (1:1, 18 mL) 16 was added nmol, 2000 µL,7719.8. 2 mM), and the resulting mixture oligonucleotide concentration = 0.5(4000 mM) Conjugate 23a. Tosolution a and solution of 21a (800 nmol) MeOH/H in MeOH/H 2(1:1, O (1:1, 18 mL) was added (4000 ◦ C in Conjugate 23a. ofoven 21a (800 nmol) 2O 18was mL) 16 16 was added (4000 nmol, 2000 2TomM), the resulting mixture (final oligonucleotide concentration = 0.5 mM) heated at 90µL, a amicrowave for 90 min.inAfterwards, MeOH removed under reduced nmol, 2000 2 mM), resulting mixture (final oligonucleotide concentration = 0.5 mM) o2 nmol, 2000 µL, andand thethe resulting mixture concentration = 0.5 mM) heated atand 90µL, C mM), in a microwave oven forby 90HPLC. min.(final Afterwards, MeOH was removed under reduced pressure the resulting crude purified HPLColigonucleotide (Figure 11): Analysis conditions, 0→ 50% B oC oin heated at 90 C in a microwave oven for 90 min. Afterwards, MeOH was removed under reduced heated at 90 a microwave oven for 90 min. Afterwards, MeOH was removed under reduced pressure resulting crude purified by HPLC. 11):(tR Analysis conditions, 0 → 50% in 30 min,and tR =the 25.9 min; purification conditions: 20 →HPLC 40%B(Figure in 30 min = 21.3 min). Yield (synthesis pressure and the resulting crude purified by HPLC. HPLC (Figure 11): Analysis conditions, 0 50% → 50% pressure and the resulting crude purified by HPLC. HPLC (Figure 11): Analysis conditions, 0 → B in 30 min, t R = 25.9 min; purification conditions: 20 → 40%B in 30 min (t R = 21.3 min). Yield (synthesis and purification): 20%. B 30 in min, 30 min, R = 25.9 min; purification conditions: 20 40%B → 40%B in min 30 min R = 21.3 min). Yield (synthesis B inand tR =t25.9 min; purification conditions: 20 → in 30 (tR =(t21.3 min). Yield (synthesis purification): 20%. and purification): 20%. and purification): 20%.

Figure purified (right) (right) conjugate conjugate 23a. 23a. Detection Detectionwavelength: wavelength: Figure11. 11.HPLC HPLC traces traces of of crude crude (left) (left) and purified Figure HPLC traces of crude (left) purified (right) conjugate Detection wavelength: 254 nm. Figure 11. 11. HPLC traces of crude (left) andand purified (right) conjugate 23a.23a. Detection wavelength: 254 nm. 254254 nm.nm.

MALDI-TOF MS 7772.5 [M − H]−−,, M for M calcd. calcd. for MALDI-TOF MS (negative (negative mode, mode,THAP/CA): THAP/CA):m/zm/z 7772.5 [M-H] −, −,M Mcalcd. calcd. for MALDI-TOF MS (negative mode, THAP/CA): m/z 7772.5 [M-H] C H BrN O P S 7767.9 for MALDI-TOF MS (negative mode, THAP/CA): m/z 7772.5 [M-H] 250 326 73 130 20 20 C250H326BrN73O130P20S20 7767.9 C 250 H 326 BrN 73 O 130 P 20 S 20 7767.9 Conjugate 23b. A solution of 21b (800 nmol) in MeOH/H O (1:1, 19 mL) (final oligonucleotide C250H326Conjugate BrN73O130P23b. 20S20 A 7767.9 solution of 21b (800 nmol) in MeOH/H22O (1:1, 19 mL) (final oligonucleotide ◦ C in Conjugate 23b. A of 21b (800 in MeOH/H 2(1:1, Ofor (1:1, 19 mL) (final oligonucleotide concentration = 40 µM) was at(800 microwave oven min. Afterwards, a solution of oCnmol) Conjugate 23b. A solution of 21b nmol) MeOH/H 2O 1990 mL) (final oligonucleotide concentration = 40 µM)solution washeated heated at90 90 inaain microwave oven for90 min. Afterwards, a solution oC in a microwave oven for 90 min. Afterwards, a solution o concentration = 40 µM) was heated at 90 16 (4000 nmol) in H O (1 mL) was added and the resulting mixture left stirring for 1 h. Finally, MeOH concentration 40 µM) was 90 added C in a and microwave oven for 90 min. 2 H of 16 (4000 =nmol) in 2O heated (1 mL) at was the resulting mixture leftAfterwards, stirring for a1solution h. Finally, of 16 (4000 nmol) inreduced H 2(1 O mL) (1 reduced mL) was added and resulting mixture left stirring for h. Finally, removed under pressure and the resulting crude purified by RP-HPLC. HPLC 12): of was 16 (4000 nmol) in H 2O was added and thethe resulting mixture left stirring 1 h.1(Figure Finally, MeOH was removed under pressure and the resulting crude purified byfor RP-HPLC. HPLC MeOH was removed under reduced pressure and the resulting crude purified by RP-HPLC. HPLC Analysis conditions, 0 → 50% B in 30 min, t = 26.7 min; purification conditions: 20 → 40% B in 30 min MeOH was removed under reduced pressure and the resulting crude purified by RP-HPLC. HPLC R (Figure 12): Analysis conditions, 0→ 50% B in 30 min, tR = 26.7 min; purification conditions: 20 → 40% (Figure 12): Analysis conditions, 0→ 50% in min, 30 min, = 26.7 min; purification conditions: 20 40% → 40% (t 8.5min min). Yield (synthesis and purification): 29%. (Figure 12): Analysis conditions, 50% B inBand 30 tR =tR26.7 min; purification conditions: 20 → Rin=30 B (t R = 8.5 min). Yield0→ (synthesis purification): 29%. in 30 min = 8.5 min). Yield (synthesis purification): 29%. B inB 30 min (tR =(tR8.5 min). Yield (synthesis andand purification): 29%.

Figure 12. HPLC traces of crude (left) and purified (right) conjugate 23b. Detection wavelength: Figure 12. HPLC traces crude (left) purified (right) conjugate 23b. Detection wavelength: Figure 12. HPLC traces of crude (left) (right) conjugate Detection wavelength: Figure 12. HPLC traces crude (left) andand purified (right) conjugate 23b.23b. Detection wavelength: min: 16. 254 nm. Left trace, tR ~of18of 254 nm. Left trace, t ~ 18 min: 16. R ~ 18 min: 16. 254 nm. Left trace, t R 254 nm. Left trace, tR ~ 18 min: 16.

Molecules 2019, 24, 579

16 of 17

MALDI-TOF MS (negative mode, THAP/CA): m/z 7758.7 [M − H]− , M calcd. C250 H326 BrN73 O130 P20 S20 7767.9.

for

3.4. Luciferase Induction Experiments Oligonucleotides conjugated to Retro 1 or its derivatives were tested in a splice correction assay using HeLa Luc 705 cells. These cells contain a firefly luciferase expression cassette interrupted by an abnormal intron from thalassemic beta-globin. As a result the cells fail to splice correctly and do not produce luciferase. However, successful intracellular delivery of a splice switching antisense oligonucleotide can correct splicing leading to luciferase expression. Thus this system provides a rapid and convenient assay for the intracellular delivery of oligonucleotides. In the current experiments the HeLa Luc 705 cells were incubated with various concentrations of oligonucleotide and then processed for luciferase induction as described [19]. In some cases there was a brief post-incubation with UNC7938, a small molecule known to enhance oligonucleotide effectiveness [15]. The cells were initially plated at 50,000 per well in 24 well tissue culture plates in DMEM (Dulbecco modified Eagles minimal essential medium) + 10% fetal bovine serum. After overnight incubation at 37 ◦ C and 5% CO2 , medium was removed and replaced with fresh complete medium containing various concentrations of the oligonucleotides. Cells were then further incubated for 16 h. In some samples this was followed by a 1 h exposure to UNC7938 followed by removal of the compound. After an additional incubation of 2 h, cells were rinsed twice with PBS and then lysed in 1/5 diluted luciferase lysis buffer (Promega, Madison, WI, USA) according to manufacturer’s directions. Luciferase activity was measured using a FLUOstar Omega microplate reader (BMG LABTECH, Cary, NC, USA) and luciferase reagents obtained from Promega. Author Contributions: Conceptualization, E.P. and A.G.; funding acquisition, E.P. and A.G.; investigation, J.A., S.M.K. and R.L.J.; writing—original draft preparation, J.A., R.L.J. and A.G.; writing—review and editing, J.A., E.P., S.M.K., R.L.J. and A.G.; visualization, J.A., R.L.J. and A.G.; supervision, R.L.J. and A.G. Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO), grant numbers CTQ2014-52658-R and CTQ2017-84779-R. Acknowledgments: We wish to acknowledge the Generalitat de Catalunya (2014SGR187 and 2017SGR391). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Sample Availability: Samples of the compounds 3, 4, 5, 8 and 13 are available from the authors. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).