This is a self-archived – parallel-published version of an original article. This version may differ from the original in pagination and typographic details. When using please cite the original. This document is the Accepted Manuscript version of a Published Work that appeared in final form in The Journal of Organic Chemistry, copyright © 2024 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.joc.4c01053 TITLE: Tuning the Solubility of Soluble Support Constructs in Liquid Phase Oligonucleotide Synthesis AUTHOR: Petja Rosenqvist, Verneri Saari, Mikko Ora, Alejandro Gimenez Molina, Andras Horvath, and Pasi Virta YEAR: 2024 DOI: 10.1021/acs.joc.4c01053 VERSION: Author’s accepted manuscript CITATION: Petja Rosenqvist, Verneri Saari, Mikko Ora, Alejandro Gimenez Molina, Andras Horvath, and Pasi Virta. The Journal of Organic Chemistry 2024, 89 (18), 13005–13015. https://doi.org/10.1021/acs.joc.4c01053 Tuning the Solubility of Soluble Support Constructs in Liquid Phase Oligonucleotide Synthesis Petja Rosenqvista,‡, Verneri Saaria,‡, Mikko Oraa, Alejandro Gimenez Molinab, Andras Horvathb, Pasi Virta*a aDepartment of Chemistry, University of Turku, 20500 Turku, Finland bJanssen Pharmaceutica N.V., 30 Turnhoutseweg, B-2340 Beerse, Belgium ABSTRACT: Solubility of the growing oligonucleotide-soluble support constructs in the liquid phase oligonucleotide syn- thesis (LPOS) is a critical parameter, which affects coupling efficiency, purity and recovery of the growing oligonucleotides during the chain elongation. In the present study, oligonucleotides have been assembled on a 4-oxoheptanedioic acid (OHDA) linker-derived tetrapodal soluble support using 5’-O-(2-methoxyprop-2-yl)-protected 2’-deoxyribonucleotide phosphoroamidite building blocks with different nucleobase protecting groups [isobutyryl (Gua), 1-butylpyrrolidin-2-yli- dene (Gua, Cyt), 2,4-dimethylbenzoyl (Ade, Cyt) and Bz (Thy)]. The solubility of the oligonucleotide-soluble support con- structs (molecular mass varying between 3-10 kDa) as models of protected tetra-, octa-, dodeca-, hexadeca- and eicosa- nucleotides was measured in different solvent systems and in potential anti-solvents. By tuning the nucleobase protecting group scheme, the solubility can be improved in aprotic organic solvent systems, while the recovery of the constructs in the precipitation, used for the isolation and purification of the growing oligonucleotide intermediates in a protic antisolvent (2-propanol), remained near quantitative. The precipitation-based yield of the protected tetrapodal oligonucleotides varied from quantitative to 90% yield. Overall yield (for di-:95%, tri-: 79-96%, tetra-: 82-88% and pentanucleotides: 68-75%) and purity of the LPOS were evaluated by RP HPLC and MS-spectroscopy of the released oligonucleotide aliquots. In addition, orthogonality of the OHDA linker was applied to release authentic protected nucleotides from the soluble supports. INTRODUCTION The growing demand for synthetic oligonucleotides, driven by their expanding application into large therapeu- tic fields, like cardiovascular diseases1,2, has challenged current industrial manufacturing3-5 that is reliant on the automated solid phase synthesis6 with limited scalability and sustainability. To make oligonucleotide synthesis more reagent efficient and process-compatible, liquid phase oligonucleotide synthesis (LPOS)7,8 has received growing attention9, including nowadays advanced technol- ogies for the large-scale production of therapeutically rele- vant oligonucleotides.10-13 The LPOS technologies aim to harness reactivity features of classical solution phase syn- thesis, applicable for real-time batch-like optimized pro- cess of the reactions, but also for facile quantitative physi- cal or mechanical isolation of the intermediate products from the reactant media. Appropriate soluble supports are utilized in LPOS, facilitating separation of the growing ol- igonucleotides from the reactants by precipitation, 10,14,15 liquid-liquid extraction12 or membrane filtration16. Soluble supports are consisted of solubility tags,10-12,17,18 appropriate branching units (star-like dendrimers),13,15,16 or their com- bination, in which the protected oligonucleotide them- selves become dominant at later steps of the synthesis. It should be realized that a significant mass portion of the protected oligonucleotides comes from the protecting groups (about 30% of the total mass in the standard DNA protecting group scheme), contributing markedly to the solubility of the construct, solvent volume of the reactions and, consequently, to the coupling and 5’-O-deprotection Figure 1. The 5’-O-MIP-protected phosphoramidite building blocks used for the LPOS (non-standard protecting groups emphasized) efficiency on these supports. This problem setting re- sembles swelling properties and suspension volumes of solid supports, which can be drastically different in the be- ginning and in the end of the biopolymer assembly due to the growing biopolymer payload. This and its role for the synthetic efficiency has extensively been studied in solid phase peptide synthesis (SPPS),19,20 but to a lesser extent in solid phase oligonucleotide synthesis (SPOS).21,22 Altered swelling is not an issue with controlled pore glass, but may affect synthetic efficiency on polystyrene supports used for SPOS.23 Solubility plays a marked role in the LPOS tech- nologies under development, including the convergent ones,10 which may need further consideration of the pro- tecting group scheme. At the same time, precipitation, fil- tration or extraction properties should result in quantita- tive isolation and purification of the growing oligonucleo- tide products from the reactant media. In the present study, a set of 5’-O-(2-methoxyprop-2-yl = MIP)-protected 2’-deoxyribonucleotide phosphoroamidite building blocks (1-6, Figure 1) were synthesized (Scheme 1) and used for LPOS on a 4-oxoheptanedioic acid (OHDA)24 linker-derived tetrapodal precipitative soluble support (Scheme 2 and 3). The solubility of the oligonucleotide-sol- uble support constructs (17 – 27) as models of protected tetra-, octa-, dodeca-, hexadeca- and eicosa-nucleotides was measured after each coupling cycle (including the cou- pling, oxidation and 5’-O-deprotection) in different solvent systems and in potential anti-solvents. The role of nucleo- base protecting group scheme [isobutyryl (Gua), 1-bu- tylpyrrolidin-2-ylidene (Gua, Cyt), 2,4-dimethylbenzoyl (Ade, Cyt) and Bz (Thy)] in the solubility and precipitation efficiency of the oligonucleotide-soluble support con- structs was examined. Aliquots of the oligonucleotides were released from the precipitated soluble supports by concentrated ammonia to verify yield, purity and authen- ticity of the oligonucleotide content. In addition, hydra- zine acetate was used for the orthogonal cleavage of the OHDA linker that released authentic protected nucleo- tides from the soluble support. RESULTS AND DISCUSSION Synthesis of the 5’-O-MIP-protected phospho- ramidite building blocks. The phosphoramidite building blocks (1-6) used in this study are described in Figure 1. MIP was selected as an alternative 5’-O-protecting group.25,26 In comparison to 4,4’-dimethoxytrityl (DMTr) group, the fast acid-catalyzed pseudo irreversible removal and the resultant volatile byproducts (acetone and metha- nol) make MIP an attractive protecting group for LPOS27, and especially for the one-pot coupling-deprotection syn- thesis cycle28 applied in the present study. N3-Bz-thymi- dine (4) and 1-butylpyrrolidin-2-ylidene (BuP) protected 2’-deoxyguanosine (5) and 2’-deoxycytidine (6) represent more lipophilic alternatives for thymidine (1)25, N-isobu- tyryl(iBu) 2’-deoxyguanosine (2) and N-2,4-dimethylben- zoyl (dmb) 2’-deoxycytidine (3) building blocks. dmb is used for Ade and Cyt due to its higher hydrolytic stability29 compared to Bz, which is beneficial for LPOS,27 and for the hydrazine acetate-mediated orthogonal cleavage of the OHDA linker (cf. below). 1 has been described previously,25 whereas syntheses of 2 - 6 are outlined in Scheme 1. As pre- viously reported, 3’-O-tert-butyldimethylsilyl (TBS)- protected nucleosides were used as key intermediates to introduce MIP selectively to the 5’-OH group of each nu- cleoside. As an example, typical procedure for N3-benzoyl- 3’-O-TBS thymidine (7)30: The 3’-O-TBS-protected nucleo- side (7) is exposed to a mixture of 2,2-methoxypropane in THF in the presence of a catalytic amount of p-TsOH. The obtained 5’-O-MIP-3’-O-TBS-protected intermediate (8) Scheme 1. Synthesis of the 5’-O-MIP-protected phosphoramidite building blocks 2-6 Conditions: i) 2,2-dimethoxypropane, THF, TsOH·H2O, for 2h at r.t.; ii) TBAF, THF, for 2h at r.t.; iii) NH3, H2O, THF, for 16h at 40°C; iv) 1-butyl-2,2-dimethoxypyrrolidine, MeOH, for 1h at r.t.; v) 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphoramidite, tetrazole, DCM, for 2h at r.t. Scheme 2. Synthesis of the tetrapodal soluble support (17) Conditions: i) 1: Me3SiCl, Et3N, DCM, for 1h at 0 → r.t.; 2: 2,4-dimethyl benzoylchloride added to the former mixture, at 0 → r.t. overnight; 3: N-methylpiperazine, Et3N added to the former mixture, r.t. overnight; 4: pyridinium fluoride added to the former mixture, for 3h at r.t.; ii) DMTrCl, Py, for 2h at r.t.; iii) 1: 4-oxoheptanedioic acid, DCC, DMAP, THF, for 16h at r.t.; 2: 19 and DBU added, and then continued overnight at r.t.; iv) N-methyl propynylamine, EDC, HOBt, DCM, for for 12h at r.t.; v) TFA, 1-dodecanethiol, DCM, for 2h at 0°C; vi) CuI, sodium ascorbate, dimethylacetamide, for 48h at r.t. was treated with TBAF to give the desired 5’-O-MIP pro- tected nucleoside (9). 5’-O-MIP protected nucleosides (10 and 13) were used as precursors for BuP-protected guano- sine and cytosine. The amide nucleobase protecting groups (Bz or iBu) were removed by concentrated ammonia and the nucleosides (11 and 14) with the exposed exocyclic amino group were then treated with 1-butyl-2,2-dimethox- ypyrrolidine, following the procedure described for 1-me- thyl-2,2-dimethoxypyrrolidine-protected nucleosides by Caruthers et al.,31 to give the desired amidine protected nu- cleosides (12 and 15). The same amidine protection led to stability issues in case of adenine,31 and the corresponding building block could not be offered. Phosphitylation of the 3’-OH group (9, 12, 1327, 15 and 1627) using 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite in the pres- ence of tetrazol gave the desired phosphoramidites 2-6. Scheme 3. Liquid phase oligonucleotide synthesis (LPOS) and yields of the oligonucleotide products Conditions: One precipitation/synthesis cycle: i) 1: Coupling: 0.095 mol L-1 of phosphoramidite (1-4, 2 eq/5’-OH group), tetrazole (2 eq/5’-OH group) in MeCN-DMF (1:1, v/v), 2h at r.t., 2: Oxidation: m-CPBA (3.8 eq / 5’-OH group), 5 min at r.t., 3: Deprotection: DCA (15% of the total volume of the reaction mixture), 15 min at r.t.; 4: Precipitation in 2-propanol. Two precipitations / synthesis cycle: ii) 1: Coupling: 0.095 mol L-1 of phosphoramidite (5 or 6, 2 eq/5’-OH group), tetrazole (2 eq/5’-OH group) in MeCN-DMF (1:1, v/v), 2h at r.t., 2: Oxidation: I2 in THF/Py/H2O added to the reaction mixture, instant at r.t.; 3: Precipitation in 2-propanol; 4: Deprotection: DCA- MeOH-DCM (5:18:27, v/v/v), 15 min, at r.t.; 5: Precipitation in 2-propanol. Table 1. Physical parameters of soluble support-oligonucleotide constructs 17, 24-33 Soluble support construct Molecular mass / g mol-1 Protecting group content, including the support / m% tetrapodal support / m% nucleobase protecting group content / m% mass concentrationa used for LPOS /g L-1 17 3023.3 67 49 18 36 24 4452.3 50 34 12 53 25 4868.7 55 31 20 58 26 6349.9 47 24 17 75 27 6314.1 47 24 16 75 28 6766.4 50 22 22 80 29 6730.5 50 22 22 80 30 7779.0 41 19 14 92 31 7743.1 41 19 13 92 32 9588.4 39 16 14 114 33 9765.0 40 15 16 116 aCorresponds to 0.012 mol L-1 solutions of 17-27. A mixture of MeCN-DMF (1:1, v/v) as a solvent. Table 2 Solubility of soluble support-oligonucleotide constructs 17, 24-33 Soluble support construct MeCN / g L-1 MeCN/DCM (1:1) / g L-1 DCM / g L-1 DCM-MeOH (1:1) / g L-1 MeCN-DMF (1:1) / g L-1 MeCN-Py (4:1) / g L-1 MeOH / g L-1 2-propanol / g L-1 MTBE / g L-1 17 0.12 29 ± 2 >300 >300 > 300 16 ± 2 0.5 ≈ 0 ≈ 0 24 0.007 1.1 0.5 >300 > 300 13 ± 1 0.06 ≈ 0 ≈ 0 25 1.2 ± 0.4 >300 >300 >300 > 300 >300 0.02 ≈ 0 ≈ 0 26 0.004 0.8 0.3 >300 > 300 10 ± 1 ≈ 0 ≈ 0 ≈ 0 27 4.4 ± 0.4 13 ± 1 0.11 >300 >300 >300 3.0 ± 0.1 ≈ 0 ≈ 0 28 26 ± 1 >300 >300 >300 >300 >300 18 ± 1 ≈ 0 ≈ 0 29 48 ± 2 >300 >300 >300 > 300 >300 32 ± 1 ≈ 0 ≈ 0 30 0.004 0.15 0.04 >300 > 300 7.0 ± 0.2 ≈ 0 ≈ 0 ≈ 0 31 1.7 ± 0.4 5.8 ± 0.9 0.06 >300 >300 130 ± 20 5.0 ± 0.9 0.003 ≈ 0 32 0.006 0.3 0.003 >300 > 300 3.0 ± 0.3 0.01 ≈ 0 ≈ 0 33 0.5 2.3 ± 0.4 0.07 >300 >300 14 ± 4 1.1 ± 0.4 0.003 ≈ 0 Note: the solubility values less than 1g L-1 and more than 300 g L-1 are given with one-digit precision without error limits. For the values more than 1 g L-1 (and less than 300 g L-1), two digits and error limits (extracted from the fitted calibration curves of the solubilities) are given. 4-oxoheptanedioic acid linker-derived tetrapodal soluble support. Codon-based SPOS32,33 and convergent LPOS10 using protected tri- and tetranucleotides as build- ing blocks may offer analytical benefits and quality im- provement for oligonucleotide end products. The key structure for the synthesis of these blockmers is an orthog- onal 3’-O-protection/linker moiety that should be selec- tively cleavable in the presence of other protecting groups. We have previously utilized a disulfide-linker34 and Q- linker35 on soluble supports for the preparation of pro- tected trinucleotide synthons in solution. In the present study, 4-oxoheptanedioic acid (OHDA) linker24 was intro- duced to the tetrapodal soluble support. The levulinoyl es- ter moiety of this linker undergoes orthogonal cleavage by hydrazine acetate, which may be applied for the release of protected nucleotides on demand, or it can be cleaved by concentrated ammonia upon the global deprotection as regular ester linker structures. Synthesis of the soluble sup- port 17, preloaded with N-dmb 2’-deoxyadenosine, is de- scribed in Scheme 2. The exocyclic amino group of 2’-de- oxyadenosine was N-dmb-protected (18), DMTr was tem- porarily introduced to the 5´-OH group (19) and the 3’-OH group of 19 was acylated with 4-oxoheptanedioic acid (20). Amide coupling with N-methyl propargylamine (21) and removal of the DMTr group gave alkyne precursor 22, which was attached to tetrapodal azide core 23 in 96% yield using Cu(I)-catalyzed click reaction. It may worth of noting that OHDA was attached to the pentaerythritol core via a secondary amide (cf. the N-methyl group) to prevent intra- molecular hemiaminal formation and subsequent prema- ture cleavage of the linker. Liquid Phase Oligonucleotide Synthesis. We have in- troduced pentaerythritol-derived soluble supports for the precipitation-based LPOS of short DNA and RNA se- quences, applying phosphoramidite,15,36,37 phos- photriester,38 and more recently stereo-controlled limo- nene-based P(V) chemistry27,39,40. In each case two precipi- tations were used to isolate the growing soluble support- bound nucleic acid intermediates: one after the coupling and the other after the 5’-deprotection. The synthesis cycle on 17 (Scheme 3) follows practically the same procedure as previously. In addition, one precipitation/coupling cycle approach was applied as follows: 2 equiv. / 5’-OH group and 0.095 mol L-1 solutions of phosphoramidites (1-4) in the presence of stoichiometric amount of tetrazole in MeCN-DMF (1:1, v/v) were used for the phosphoramidite coupling (2h, at r.t.), followed by oxidation with m-chloro- perbenzoic acid (mCPBA, 3.8 equiv / 5’-OH group, 5 min, at r.t.). After coupling, dichloro acetic acid (DCA) was added to the reaction mixture (15% DCA of the total vol- ume, for 15 min at r.t.) to deprotect the 5’-O-MIP group and the oligonucleotide-soluble support construct (24, 26, 28, 30 and 32) was precipitated in 2-propanol. Precipitation yields of 24, 26, 28, 30 and 32 varied from 96% to quantita- tive (Scheme 3). Recently, a careful impurity analysis has been done in convergent LPOS using phosphoramidite chemistry, in which by-products of partial premature cy- anoethyl removal (de-CE) have been observed (without an obvious exposure to a base) and considered as noncritical impurities.10 MS(ESI-TOF) analysis of the precipitated con- structs (24, 26, 28, 30, 32) verified their authenticity in vir- tually intact form, although partial de-CE could be de- tected in the end of the assembly (cf. Figure S44). Char- acterization of the released nucleotides afforded more de- tailed analysis of the synthesis. Overall yield (for di-: 95%, tri-: 79 and 96%, tetra-: 88% and pentanucleotide: 75%, Scheme 3), purity (97%, 89%, 93%, 87% and 73% for di-, tri-, tetra- and pentanucleotides, respectively) and authen- ticity of the nucleotides, released from aliquots of the pre- cipitates by concentrated ammonia, were determined by HPLC (Scheme S1), UV-absorbance at λ = 260 nm and MS- spectroscopy (Figures S45, S46, S48, S49). In case of phos- phoramidites 5 and 6, and soluble support-oligonucleotide constructs 27, 29, 31 and 33, two-precipitation / coupling cycle and iodine-oxidation (due to the susceptibility of PuB to mCPBA) were applied. After coupling, 0.2 M I2 in THF/Py/H2O (5:4:1 v/v/v) was added to the mixture to oxi- dize the phosphite triester intermediate and the product (5’-O-MIP protected 27, 29, 31 and 33) was precipitated in 2-propanol. The precipitate was exposed to a mixture of DCA-MeOH-DCM (5:18:27, v/v/v, 15 min, at r.t.) to remove MIP and the product (27, 29, 31 or 33) was precipitated in 2-propanol. Yields of the oligonucleotide-soluble support constructs over two precipitations varied from 90 to 94% (Scheme 3). Overall yield (for tri-: 83% and 92%, tetra-: 82% and pentanucleotide: 68%, Scheme 3), purity (for tri-: 91% and 85%, for tetra-: 81% and for pentanucleotide: 70%) and authenticity (Scheme S1, Figures S45-S47 and S50) of the released oligonucleotides were determined as above. The one-pot synthesis cycle, consisted of sequential cou- pling, oxidation, and deprotection, followed by a single precipitation, may make the synthesis operationally sim- pler and improve the overall yield of the oligonucleotides.28 The two precipitations /synthesis cycle-technique is more labor intensive and may cause unnecessary product losses due to the extra precipitations. However, the compatibility of the reagents (the latter steps should accept the remain- ing reagent traces left from the previous ones) used for LPOS and isolation and precipitation/purification effi- ciency of the of the growing oligonucleotides in the reac- tant media may affect the superiority of these techniques. Solubility of the soluble support-oligonucleotide constructs. The molecular mass of the soluble support-ol- igonucleotide constructs (17, 24-33) increases from 3023 g mol-1 (17) to 9765 g mol-1 (33) during the LPOS (Table 1). At the same time, the total mass content (m%) of the protect- ing groups decreases from 67 m% to 40 m%, and the con- tent of the tetrapodal support from 49 m% to 15 m% (Fig- ure S51). The nucleobase protecting group content varies between 12 m% and 22 m%. When the length of the oligo- nucleotide increases, the solubility of the constructs 17, 24- 33 decreases, as expected. The mass concentration of 17, 24-33 used for LPOS had to increase from 36 g L-1 to 116 g L- 1 as the oligonucleotide chain grew (corresponds to 0.012 mol L-1 solutions of the constructs 17, 24-33, needed for 0.095 mol L-1 solutions of phosphoramidite building blocks 1-6, 2 equiv/5’-OH group). The solubilities of the con- structs (17, 24-33) in various solvents and solvent mixtures are presented in table 2. The solubilities were measured in aprotic solvents acetonitrile (MeCN) and dichloromethane (DCM), in solvent mixtures of MeCN/DCM (1:1, v/v), MeCN/DMF (1:1, v/v) and pyridine/MeCN (1:1, v/v), as well as in potential antisolvents methanol, 2-propanol and me- thyl tert-butyl ether (MTBE). A solubility of 300 g L-1 has been set to represent an upper limit value (a value that is clearly more than needed for synthetic purposes). This was obtained for all constructs (17, 24-33) in mixtures of DCM- MeOH (1:1, v/v) and MeCN-DMF (1:1, v/v), used as depro- tection and coupling solvent systems in the present study. Each of the constructs (17, 24-33) had modest solubility in MeCN,41,42 the common solvent choice for standard phosphoramidite coupling. In general, with dmb- and iBu- protected nucleobases and without N3-protection of Thy (24, 26, 30 and 32), modest solubility can be observed also in DCM and in a mixture of MeCN-DCM (1:1, v/v), used fre- quently to dissolve oligonucleotide segments and trinucle- otide blockmers for the coupling.10,35,43- Examination of the results show the relationship between the nucleobase pro- tecting groups and the solubility. By introducing of more lipophilic 1-butylpyrrolidin-2-ylidene (BuP) protection for dC (27, 31) and dG (33), solubility in aforementioned apro- tic solvent systems increased, but remained still low, to po- tentially hamper the reactivity on the soluble support-oli- gonucleotide constructs in these solutions (cf. the required mass concentration of the constructs 17, 24-33 in Table 1). Interestingly, 27 and 31 showed good solubility (Figure S52), but 33 did not sufficient one (14 g L-1, 0.014 mol L-1), in a mixture of MeCN-Py (4:1, v/v), which is a readily volatile and non-halogenated solvent alternative for the phospho- ramidite coupling (compared to mixtures of DMF and DCM, respectively). The N3- protection of Thy had the strongest impact, resulting in full solubility of constructs 28 and 29 in DCM, MeCN-DCM (1:1, v/v) and MeCN-Py (4:1, v/v)(Figure S53). Benzoyl at N3 however, is susceptible to premature cleavage, which dilutes its value for further ap- plicability. Further research could evaluate N3-pivalo- yloxymethyl (Pom) protected thymine40 as a solubility im- proving group. Favourably, the solubility in antisolvents 2- propanol and MTBE remained negligible in all cases. Solu- bility in MeOH increased when the more lipophilic pro- tecting groups were introduced to the oligonucleotides (tetrapodal dodecanucleotides 26-29: from 0 to 32 g L-1), but similar trend cannot be seen in 2-propanol and MTBE. As we have previously noticed27, 2-propanol is the superior antisolvent in LPOS, resulting in quantitative precipitation and efficient removal of reagent traces. Orthogonal cleavage of protected nucleotides from the soluble supports. To demonstrate the applicability of hydrazine acetate-mediated orthogonal cleavage of the OHDA linker, two protected trinucleotides 5´-O-MIP- dC(dmb)-T-dA(dmb)-OH (34) and 5’-O-MIP-T-dG(BuP)- dA(dmb)-OH (35) were synthesized following the LPOS protocols above (Scheme 4). The trinucleotide-soluble support constructs (34 and 35) were obtained in 96% and 92% overall yields, respectively. The precipitates were ex- posed to a mixture of 0.1 mol L-1 hydrazine acetate [5 eq, hydrazine hydrate-acetic acid-pyridine (0.025:1:4, v/v/v), 1h at r.t.] (RP HPLC monitoring of the orthogonal cleavage- shown in Figure S54), followed by extraction between DCM and saturated NaHCO3 and elution through a short silica gel column. Protected trinucleotides were obtained in 71% (36) and 65% (37) yields, corresponding to 68% an 60% overall yields from 17, respectively. Scheme 4. Cleavage of protected trinucleotides from the soluble support. Conditions: i) NH2NH2·H2O-AcOH-Py (0.025:1:4, v/v/v), 1h at r.t. CONCLUSION 5’-O-(2-methoxyprop-2-yl = MIP)-protected 2’-deoxyri- bonucleotide phosphoroamidite building blocks, with dif- ferent nucleobase protecting groups, were synthesized and used for LPOS on a 4-oxoheptanedioic acid (OHDA)24 linker-derived tetrapodal soluble support. The solubility of the oligonucleotide-soluble support constructs was meas- ured in different solvent systems and in potential anti-sol- vents. The solubility of the constructs (molecular mass var- ying between 3-10 kDa) decreased as the oligonucleotide chain grew, and became in some cases low in most com- mon solvent choices (MeCN and mixtures of MeCN and DCM) used for the phosphoramidite-based chain elonga- tion of oligonucleotides. The nucleobase protecting group scheme [isobutyryl (Gua), 1-butylpyrrolidin-2-ylidene (Gua, Cyt), 2,4-dimethylbenzoyl (Ade, Cyt) and Bz (Thy)] may be tuned to improve the solubility of the constructs in organic solvent systems, including also alternative solvent choices suitable for the phosphoramidite coupling (a mix- ture of MeCN and pyridine). At the same time, the solubil- ity of the constructs in 2-propanol remained unchanged and negligible to achieve efficient precipitation and purifi- cation of the constructs. The results obtained here high- lights the importance of the solubility of the growing pro- tected oligonucleotides in coupling conditions, which can be challenging at the later stage of the chain elongation, and that LPOS strategies can benefit from further protect- ing group optimization. With an appropriate choice of the nucleobase protecting groups, alternative potentially greener reaction solvents can be accessed that will further increase the attractiveness of the LPOS methodologies. The described challenges in solubility (note the decreasing trend when the length of the oligonucleotide increases) also implies that the branched or star-like soluble sup- ports, such as the pentaerythritol-derived one used in our studies, may be suitable for the preparation of short pro- tected oligonucleotide synthons, but efficient synthesis of longer oligonucleotides in solution would favour linear as- sembly of these synthons applying for example additional solubility determining groups (SDGs).10 The applicability of the OHDA-linker24 in LPOS for the preparation of pro- tected trinucleotides, as precursors for such synthons10,32,33 has been demonstrated and proven to be competitive with our previously published procedures, applying Q- and di- sulfide linkers.34,35 EXPERIMENTAL SECTION General Methods. 1H, 13C and 31P NMR spectra were rec- orded on Bruker Avance 500MHz and 600 MHz instru- ments. For the RP HPLC analysis, an analytical C18 column (4.6 × 250 mm, 5 μm, flow rate 1 mL/min, detection at λ = 260 nm) and a gradient elution of an aqueous 50 mM tri- ethylammonium acetate and MeCN were used. For the synthesis, DCM and DMF were dried over 4 Å molecular sieves and MeCN and MeOH over 3 Å molecular sieves. 1- butyl-2,2-dimethoxypyrrolidine was prepared from 1-bu- tylpyrrolidin-2-one following the procedure described for 1-methyl-2,2-dimethoxypyrrolidine by Caruthers et al.31: 1- butylpyrrolidin-2-one (50 g, 350 mmol) was added to dime- thyl sulfate (67 g, 533.1 mmol, 1.5 eq.) at 25 ℃. After addi- tion, the reaction was warmed to 90 ℃ on an oil bath and stirred for 2 hours. Then the mixture was cooled down to - 10 ℃. A solution of sodium methoxide (25 g, 0.46 mmol, 1.3 eq.) in MeOH (250 mL) was added at -10 ℃ and the mixture was stirred at 25 ℃ for one hour. The solution was filtered and the filtrate was concentrated to dryness under reduced pressure. Then MTBE (250 mL) was added to the conden- sate, the mixture was stirred at 25 ℃ for 2 hours, filtered and evaporated to give crude 1-butyl-2,2-dimethoxypyrrol- idine (50 g) as brown yellow oil which was used as such. Compounds 1, 7, 10, 13 and 16 were prepared by previously reported methods affording products with spectroscopic data matching those described in literature. 25,27,30 Caution! Hydrazine hydrate is acutely toxic and it has long lasting effects to aquatic life. All manipulations were performed on the smallest practical scale. 1-Hydroxyben- zotriazole show explosive properties when heated or me- chanically stimulated and should be handled with care. Di- chloroacetic acid is corrosive and show reproductive tox- icity and should be handled with caution. Tetrazole pre- sents a risk of explosion by shock, friction, or fire which was controlled using dilute commercial solutions. N3-Benzoyl-5’-O-(2-methoxypropane-2-yl)-2’-deoxy- thymidine (9): To a solution of 730 (100 g, 217 mmol) in 2,2- dimethoxypropane (267 mL) and THF (1000 mL), TsOH·H2O (8.3 g, 43 mmol, 0.2 eq.) was added. The reac- tion was stirred 2 hours at r.t., quenched by addition of Et3N (50 mL) and concentrated to dryness. The residue (8) was dissolved in THF (290 mL) and TBAF (114 g, 434 mmol, 2.0 eq.) was added. The mixture was stirred for 2 hours at r.t., concentrated to dryness and re-dissolved in DCM (500 mL). The mixture was washed with water three times, dried over Na2SO4, and concentrated to dryness to give a crude product. The crude was purified by silica gel chro- matography (ethyl acetate/n-heptane, 1:8 → 1:4, v/v) to give 9 as white solid (45 g, yield: 50%). 1H NMR (500 MHz, CDCl3): δ 7.92 (dd, J = 8.4, 1.2 Hz, 2H), 7.69 (d, J = 1.1 Hz, 1H), 7.67 – 7.60 (m, 1H), 7.52 – 7.45 (m, 2H), 6.34 (t, J = 6.6 Hz, 1H), 4.48 (dd, J = 5.7, 2.9 Hz, 1H), 4.09 (q, J = 3.0 Hz, 1H), 3.70 (dd, J = 10.8, 3.1 Hz, 1H), 3.65 (dd, J = 10.8, 3.0 Hz, 1H), 3.24 (s, 3H), 2.35 (ddd, J = 13.5, 6.1, 3.4 Hz, 1H), 2.21 (dd, J = 13.8, 6.7 Hz, 2H), 1.97 (d, J = 1.1 Hz, 3H), 1.42 (s, 3H), 1.41 (s, 3H). 13C{1H} NMR (126 MHz, CDCl3): δ 169.0, 163.0, 149.3, 136.0, 135.1, 131.5, 130.4 (2 × C), 129.2 (2 × C), 110.5, 100.4, 86.1, 85.2, 71.9, 60.8, 48.7, 40.9, 24.4, 24.3, 12.4. HRMS (ESI): m/z [M+H]+ calcd for C21H27N2O7+ 419.1818, found 419.1776. N3-Benzoyl-5'-O-(2-methoxypropane)-2'-deoxythymidine- 3'-O-yl cyanoethyl N,N-diisopropylaminophosphoramidite (4): To a solution of 9 (50 g, 120 mmol) in DCM (500 mL), 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodia- midite (43 g, 140 mmol, 1.2 eq.) and tetrazole (6.7 g, 96 mmol, 0.8 eq.) were added successively. The reaction was stirred for 2 hours at r.t. and quenched by addition of sat- urated aqueous NaHCO3 (400 mL). The organic phase was separated, dried over Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography (ethyl acetate/n-heptane, 1:8 → 1:2, v/v) to give 4 as white solid (45 g, yield: 61%). 1H NMR (500 MHz, CDCl3): δ 7.96 – 7.83 (m, 2H), 7.70 (d, J = 1.0 Hz, 0.55H), 7.66 (d, J = 1.0 Hz, 0.45H), 7.59 (q, J = 7.3 Hz, 1H), 7.44 (m, 2H), 6.34 (dd, J = 10.0, 3.6 Hz, 1H), δ 4.57 – 4.50 (m, 1H), 4.25 (d, J = 2.1 Hz, 0.55H), 4.16 (d, J = 2.3 Hz, 0.45H), 3.79 (m, 1H), 3.72 – 3.52 (m, 5H), 3.21 (m, 3H), 2.57 (m, 2H), 2.47 (ddd, J = 13.5, 5.9, 2.8 Hz, 0.45H), 2.41 (ddd, J = 13.2, 5.7, 2.4 Hz, 0.55H), 2.26 – 2.13 (m, 1H), 1.93 (s, 3H), 1.38 (t, J = 3.5 Hz, 6H), 1.13 (d, J = 6.9 Hz, 12H); 13C{1H} NMR (126 MHz, CDCl3) (note: dia- stereomers are observed): δ 169.0, 162.8, 149.3 (d, JC-P = 3.8 Hz), 135.6, 135.5, 134.9, 131.6, 130.4 (2 × C), 129.1 (2 × C), 117.6 (d, JC-P = 11.3 Hz), 110.7 (d, JC-P = 7.6 Hz), 100.4, 85.6 (d, JC-P = 3.8 Hz), 85.2 (d, JC-P = 6.3 Hz), 74.1 (d, JC-P = 8.8 Hz), 60.8, 58.0 (d, JC-P = 18.9 Hz), 48.8 (d, JC-P = 2.5 Hz), 43.3 (d, JC-P = 12.6 Hz), 40.0 (d, JC-P = 7.6 Hz), 24.5, 24.5, 24.4, 24.4, 24.4, 24.4, 20.4 (d, JC-P = 7.6 Hz) & 20.3 (d, JC-P = 7.6 Hz), 12.4. 31P NMR (203 MHz, CDCl3): δ 148.55, 148.38. HRMS (ESI): m/z [M+H]+ calcd for C30H44N4O8P+ 619.2897, found 619.2912. N4-[(1-butylpyrrolidine-2-ylidene)amino]-5'-O-(2-methox- ypropane)-2'-deoxycytidine (12): To a stirring solution of 1025 (189 g, 469 mmol) in THF (950 mL), aqueous 25% am- monia (0.95 L) was added. The solution was stirred at 40 oC on an oil bath for 16 hours and concentrated under re- duced pressure. The residue was precipitated in acetoni- trile to yield 11 as white solid (83 g, 59%, HRMS (ESI): m/z [M+H]+ calcd for C13H22N3O5+ 300.1559, found 300.1558 M+H+). The precipitate (11, 83 g, 280 mmol) was dissolved in MeOH (830 mL) and 1-butyl-2,2-dimethoxypyrrolidine (125 g, 666 mmol, 2.4 eq.) was added in one portion. After stirring for 1 hour at r.t., the mixture was concentrated to dryness. The residue was purified by silica gel chromatog- raphy (DCM/n-heptane, 2:1, v/v → DCM-MeOH, 20:1, v/v) to give 12 as white solid (84 g, 71%). 1H NMR (500 MHz, CDCl3): δ 8.04 (d, J = 7.3 Hz, 1H), 6.38 (t, J = 6.1 Hz, 1H), 5.97 (d, J = 7.3 Hz, 1H), 4.59 (s, 1H), 4.43 (dd, J = 10.1, 4.6 Hz, 1H), 4.16 (dd, J = 7.2, 3.3 Hz, 1H), 3.71 (dd, J = 10.6, 3.1 Hz, 1H), 3.63 (dd, J = 10.6, 3.3 Hz, 1H), 3.54 – 3.41 (m, 4H), 3.21 (s, 3H), 3.20 – 3.05 (m, 2H), 2.61 (ddd, J = 13.5, 6.1, 4.8 Hz, 1H), 2.14 (m, 1H), 2.05 – 1.97 (m, 2H), 1.56 (m, 2H), 1.39 – 1.27 (m, 8H), 0.93 (t, J = 7.4 Hz, 3H). 13C{1H} NMR (126 MHz, CDCl3): δ 172.2, 168.8, 156.8, 140.5, 103.1, 100.3, 86.6, 85.9, 71.3, 60.7, 49.4, 48.8, 44.4, 42.0, 31.0, 29.1, 24.4, 20.1 (2 × C), 19.9, 13.8. HRMS (ESI): m/z [M+H]+ calcd for C21H35N4O5+ 423.2607, found 423.2599. N4-[(1-butylpyrrolidine-2-ylidene)amino]-5'-O-(2-meth- oxypropane)-2'-deoxycytidine-3'-O-yl cyanoethyl N,N- diisopropylphosphoramidite (6): To a stirring solution of 12 (55 g, 130 mmol) in DCM (550 mL), tetrazole (7.3 g, 104 mmol, 0.8 eq.) and 2-cyanoethyl N,N,N',N'-tetrai- sopropylphosphorodiamidite (45 g, 150 mmol, 1.2 eq.) were added. After stirring for 2 hours at r.t., the mixture was washed twice with 5% aqueous NaHCO3 and brine. The or- ganic phase was separated, dried over Na2SO4 and concen- trated under reduced pressure. The residue was purified by silica gel chromatography (DCM) and preparative isocratic RP HPLC (acetonitrile-H2O, 95:5, v/v) to give 6 as colorless oil (30 g, 37%). 1H NMR (500 MHz, CDCl3): δ 7.90 (d, J = 7.3 Hz, 0.55H), 7.86 (d, J = 7.3 Hz, 0.45H), 6.33 – 6.13 (m, 1H), 5.83 (d, J = 7.3 Hz, 1H), 4.53 – 4.31 (m, 1H), 4.08 (d, J = 3.5 Hz, 0.55H), 4.04 (d, J = 3.4 Hz, 0.45H), 3.75 – 3.69 (m, 1H), 3.67 – 3.61 (m, 1.55H), 3.58 (dd, J = 10.7, 3.1 Hz, 0.45H), 3.54 – 3.43 (m, 3H), 3.36 (t, J = 7.4 Hz, 2H), 3.31 (t, J = 7.1 Hz, 2H), 3.09 (d, J = 7.1 Hz, 3H), 3.03 (t, J = 7.9 Hz, 2H), 2.54 – 2.50 (m, 2H), 2.50 – 2.43 (m, 1H), 2.11 – 2.02 (m, 1H), 1.93 – 1.85 (m, 3H), 1.48 – 1.39 (m, 2H), 1.27 – 1.23 (m, 6H), 1.22 – 1.17 (m, 2H), 1.09 – 1.03 (m, 12H), 0.80 (t, J = 7.4 Hz, 3H). 13C{1H} NMR (126 MHz, CDCl3) (note: diastereomers are ob- served): δ 172.0, 168.2 (d, JC-P = 3.8 Hz), 156.2 (d, JC-P = 1.3 Hz), 140.0 (d, JC-P = 3.8 Hz), 117.4 (d, JC-P = 5.0 Hz), 102.7 (d, JC-P = 8.8 Hz), 100.1 (d, JC-P = 1.3 Hz), 86.2, 84.9 (d, JC-P = 3.8 Hz) & 84.7, 84.7 (d, JC-P = 3.8 Hz), 72.9 (d, JC-P = 3.8 Hz), 72.3 (d, JC- P = 7.6 Hz), 60.0 (d, JC-P = 6.3 Hz), 58.2 (d, JC-P = 5.0 Hz), 58.1 (d, JC-P = 6.3 Hz), 49.1, 48.6 (d, JC-P = 5.0 Hz), 44.1, 43.1 (d, JC- P = 3.8 Hz) & 43.0 (d, JC-P = 3.8 Hz), 40.7 (d, JC-P = 5.0 Hz) & 40.6 (d, JC-P = 5.0 Hz), 30.6 (d, JC-P = 5.0 Hz), 28.9, 24.4, 24.3, 24.2, 24.1, 24.1, 24.0, 20.2, 20.2 (d, JC-P = 2.5 Hz), 20.1, 19.9, 19.6, 13.5. 31P NMR (203 MHz, CDCl3): δ 148.32 (s), 148.14 (s). HRMS (ESI): m/z [M+H]+ calcd for C30H52N6O6P+ 623.3686, found 623.3655. N2-[(1-butylpyrrolidine-2-ylidene)amino]-5'-O-(2-methox- ypropane-2-yl)-2'-deoxyguanosine (15): To a solution of 1327 (80 g, 200 mmol) in MeOH (800 mL), aqueous 25% ammo- nia (800 mL) was added . The mixture was stirred at 45 ℃ on an oil bath for 20 hours. The reaction solution was con- centrated under reduced pressure to give crude 14 (45 g, HRMS (ESI): m/z [M+H]+ calcd for C14H22N5O5+ 340.1621, found 340.1588) as a white solid which was used for next step directly. To a solution of crude 14 (45 g, 130 mmol) in MeOH (450 mL), 1-butyl-2,2-dimethoxypyrrolidine (50 g) was added and the reaction was stirred at r.t. for 30 min. DCM (450 mL) was added and the mixture was washed with water. The organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pres- sure. The residue was purified by preparative RP-HPLC (CH3CN/H2O, 65/35 → 85/15, v/v) to give 15 as white solid (20 g, yield: 22% over two steps). 1H NMR (500 MHz, DMSO-d6): δ 11.10 (s, 1H), 7.98 (s, 1H), 6.20 (t, J = 6.7 Hz, 1H), 5.38 (d, J = 4.1 Hz, 1H), 4.36 (m, 1H), 3.91 (dd, J = 8.6, 4.4 Hz, 1H), 3.55-3.38 (m, 6H), 3.06-2.96 (m, 5H), 2.66 (m, 1H), 2.27 (m, 1H), 2.05-1.93 (m, 2H), 1.60-1.48 (m, 2H), 1.30 (m, 2H), 1.25 (s, 6H), 0.91 (t, J = 7.4 Hz, 3H). 13C{1H} NMR (126 MHz, DMSO-d6): δ 168.0, 157.8, 156.7, 149.8, 136.4, 119.2, 99.6, 85.6, 82.8, 70.9, 61.1, 48.6, 47.8, 43.6, 40.0 (over- lap), 30.7, 28.4, 24.1, 24.1, 19.6, 19.4, 13.6. HRMS (ESI): m/z [M+H]+ calcd for C22H35N6O5+ 463.2669, found 463.2662. N2-[(1-butylpyrrolidine-2-ylidene)amino]-5'-O-(2-meth- oxypropane-2-yl)-2'-deoxyguanosine-3’-O-yl cyanoethyl N,N-diisopropylphosphoramidite (5): To a solution of 15 (20 g, 43 mmol) in DCM (200 mL), 2-cyanoethyl N,N,N',N'- tetraisopropylphosphorodiamidite (16 g, 52 mmol) and te- trazole (2.4 g, 34 mmol) were added under nitrogen. After stirring for 2 hours at r.t., the reaction was quenched with saturated NaHCO3 (200 mL) and the organic phase was separated, dried and evaporated. The crude product was purified by silica gel column chromatography (EtOAc/Heptane, 1:2 → 1:1, v/v) to give 5 as white solid (14 g, yield: 49%). 1H NMR (600 MHz, CDCl3): δ 9.19 (s, 0.5H), 9.18 (s, 0.5H), 7.83 (s, 0.5H), 7.82 (s, 0.5H), 6.23 (t, J = 6.6 Hz, 1H), 4.60-4.52 (m, 1H), 4.20 (d, J = 2.4 Hz, 0.5H), 4.13 (d, J = 2.7 Hz, 0.5H), 3.82-3.74 (m, 1H), 3.73-3.64 (m, 1H), 3.60-3.45 (m, 4H), 3.44-3.33 (m, 4H), 3.15-3.01 (m, 5H), 2.62- 2.54 (m, 2H), 2.54-2.39 (m, 2H), 2.05-1.90 (m, 2H), 1.51-1.43 (m, 2H), 1.35-1.18 (m, 8H), 1.11 (s, 6H), 1.10 (s, 6H), 0.83 (t, J = 7.4 Hz, 3H); 13C{1H} NMR (126 MHz, CDCl3) (note: dia- stereomers are observed): δ 169.2 (d, JC-P = 5.0 Hz), 158.0, 156.3, 150.3, 136.0 (d, JC-P = 6.3 Hz), 119.9, 117.5, 100.5 (d, JC-P = 1.3 Hz), 85.6 (d, JC-P = 7.6 Hz) & 85.6 (d, JC-P = 7.6 Hz), 83.5, 74.1 (d, JC-P = 16.4 Hz), 60.8 (d, JC-P = 13.9 Hz), 58.6 (d, JC-P = 12.6 Hz), 58.4(d, JC-P = 12.6 Hz), 49.3, 48.9 (d, JC-P = 1.3 Hz), 44.5, 43.5(d, JC-P = 12.6 Hz) & 43.4 (d, JC-P = 12.6 Hz), 40.8 (d, JC-P = 16.4 Hz) & 40.7 (d, JC-P = 16.4 Hz), 31.7, 29.1, 24.8, 24.7, 24.6, 24.6, 24.5, 24.4, 20.6 (d, JC-P = 16.4 Hz), 20.2, 19.9 (d, JC- P = 16.4 Hz), 13.9; 31P NMR (243 MHz, CDCl3): δ 148.46 (s), 148.31 (s). HRMS (ESI): m/z [M+H]+ calcd for C31H52N8O6P+ 663.3747, found 663.3712. N2-isobutyryl-5'-O-(2-methoxypropane)-2'-deoxyguano- sine-3'-O-yl cyanoethyl N,N-diisopropylphosphoramidite (2). To a solution of 13 (30 g, 73.3 mmol) in DCM (300 mL), 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (26.5 g, 87.9 mmol, 1.2 eq.) and tetrazole (4.11 g, 58.6 mmol, 0.8 eq.) were added under nitrogen. After stirring for 60 minutes at r.t., the reaction was quenched by addition of saturated aqueous NaHCO3 (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography (EtOAc/Heptane, 1:2 → 3:1, v/v) to give 2 as white solid (29.3 g, yield: 65.6 %). 1H NMR (500 MHz, CDCl3): δ 12.18 (s, 1H), 9.82 (s, 0.5H), 9.76 (s, 0.5H), 7.98 (s, 0.5H), 7.95 (s, 0.5H), 6.25-6.21 (m, 1H), 4.78-4.73 (m, 0.5H), 4.69-4.64 (m, 0.5H), 4.26-4.21 (m, 1H), 3.94-3.84 (m, 1.5H), 3.81-3.71 (m, 1.5H), 3.67-3.55 (m, 3H), 3.16(s, 1.5H), 3.15 (s, 1.5H), 2.82-2.68(m, 4.5H), 2.57-2.52 (m, 0.5H), 1.37 (s, 3H), 1.34 (d, J = 2.4 Hz, 3H), 1.24-1.17 (m, 18H). 13C{1H} NMR (126 MHz, CDCl3): δ 179.5 & 179.4, 155.8 & 155.8, 148.0 (d, J = 11.3 Hz), 147.8 (d, J = 10.8 Hz), 137.6 & 137.2, 121.5 & 121.3, 117.8 (d, J = 5.4 Hz), 100.5 & 100.4, 85.5 (d, J = 11.3 Hz) & 85.5, 84.7 & 84.2, 74.1 (d, J = 17.6 Hz) & 73.3 (d, J = 15.1 Hz), 60.8 & 60.6, 58.0 (d, J = 10.1 Hz) & 57.9 (d, J = 10.1 Hz), 48.6, 48.6, 43.3 & 43.2, 40.1 (d, J = 2.5 Hz) & 39.7 (d, J = 3.8 Hz), 36.0 & 35.9, 24.6, 24.5, 24.5, 24.4, 24.3, 24.2, 20.5 (d, J = 2.5 Hz) & 20.4 (d, J = 3.8 Hz), 19.0, 18.9 (d, J = 3.8 Hz). 31P NMR (203 MHz, CDCl3): δ 148.52 (s), 147.79 (s). HRMS (ESI): m/z [M+H]+ calcd for C27H45N7O7P+ 610.3118, found 610.3083. N4-(2,4-dimethylbenzoyl)-5'-O-(2-methoxypropane)-2'- deoxycytidine-3’-O-yl cyanoethyl N,N-diisopropylphospho- ramidite (3): To a solution of 16 (70 g, 160 mmol) in DCM (700 mL), 2-cyanoethyl N,N,N',N'-tetrai- sopropylphosphorodiamidite (59 g, 190 mmol, 1.2 eq.) and tetrazole (9.1 g, 130 mmol, 0.8 eq.) were added. After stir- ring for 2 hours at r.t., the reaction mixture was washed with 5% NaHCO3 aqueous solution and 10% NaCl aqueous solution successively. The organic phase was separated, dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by silica gel chromatography (EtOAc/n-heptane, 1:4 → 1:1, v/v) to give 3 as white solid (56 g, yield: 54.6%). 1H NMR (500 MHz, CDCl3): δ 8.73 (s, 1H), 8.46 (dd, J = 12.1, 7.5 Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 7.05 (d, J = 7.9 Hz, 1H), 6.27 (m, 1H), 4.57 – 4.47 (m, 1H), 4.31 (d, J = 2.9 Hz, 0.5H), 4.25 (d, J = 3.0 Hz, 0.5H), 3.91 – 3.82 (m, 1H), 3.79 – 3.73 (m, 2H), 3.71 – 3.55 (m, 3H), 3.24 (d, J = 5.6 Hz, 3H), 2.77 – 2.68 (m, 1H), 2.65 (t, J = 6.2 Hz, 2H), 2.49 (s, 3H), 2.35 (s, 3H), 2.26 – 2.18 (m, 1H), 1.48 – 1.33 (m, 6H), 1.32 – 1.08 (m, 12H). 13C{1H} NMR (126 MHz, CDCl3) (note: diastereomers are ob- served): δ 168.6, 162.1 (d, JC-P = 3.8 Hz), 154.9, 144.7, 142.0, 137.7, 132.6, 131.1, 127.2, 126.7, 117.5 (d, JC-P = 6.3 Hz), 100.3, 95.6, 87.1 (d, JC-P = 5.0 Hz), 85.7 (d, JC-P = 3.8 Hz) & 85.5 (d, JC-P = 3.8 Hz), 72.9 (d, JC-P = 6.3 Hz) & 72.5 (d, JC-P = 16.4 Hz), 59.8, 58.3 (d, JC-P = 3.8 Hz) & 58.1 (d, JC-P = 3.8 Hz), 48.9 (d, JC-P = 1.3 Hz), 43.3 (d, JC-P = 2.5 Hz) & 43.2 (d, JC-P = 2.5 Hz), 41.2 (d, JC-P = 7.6 Hz) & 41.0 (d, JC-P = 7.6 Hz), 24.6, 24.5, 24.5, 24.5, 24.4, 24.3, 21.3, 20.3 (d, JC-P = 7.6 Hz), 20.1. 31P NMR (203MHz, CDCl3): δ 148.63 (s), 148.55 (s). HRMS (ESI): m/z [M+H]+ calcd for C31H47N5O7P+ 632.3213, found 632.3210. 7-[N6-(2,4-dimethylbenzoyl)-2’-deoxyadenosine (18). To a solution of 2’-deoxyadenosine (89 g, 350 mmol) in DCM (890 mL), triethylamine (254 g, 2.51 mol, 7.1 eq.) and chlo- rotrimethylsilane (121 g, 1,11 mol, 3.1 eq.) were added at 0 oC. After stirring at 25 oC for one hour, 2,4-dimethylbenzoyl chloride (149 g, 886 mmol, 2.5 eq.) was added at 0 oC, and the mixture was stirred at 25 oC for 12 hours. Then, the mix- ture was washed with water (twice), the organic phase was separated, dried over Na2SO4, and filtered to give a solution which was used for the next step directly. N-methyl piper- azine (35 g, 0.34 mol, 0.97 eq.) and triethylamine (65 g, 0.64 mol, 1.8 eq.) were added and the mixture was stirred at 25 oC overnight. Pyridinium fluoride (70%, 32 mL) was added, and the mixture was stirred at 25 oC for 3 hours. The mix- ture was concentrated and the crude oil was purified by sil- ica gel column chromatography (DCM/MeOH, 50:1, v/v) to give 42.0 g of 18 (31%) as yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.69 (s, 1H), 8.68 (s, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.13 (s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.48 (t, J = 6.8 Hz, 1H), 5.36 (d, J = 4.2 Hz, 1H), 5.02 (t, J = 5.6 Hz, 1H), 4.53 – 4.38 (m, 1H), 3.91 (dd, J = 7.5, 4.5 Hz, 1H), 3.68 – 3.61 (m, 1H), 3.57 – 3.52 (m, 1H), 2.84 – 2.75 (m, 1H), 2.42 (s, 3H), 2.39 – 2.34 (m, 1H), 2.33 (s, 3H). 13C{1H} NMR (126 MHz, DMSO-d6) δ 167.82, 151.78, 151.54, 150.04, 142.93, 140.11, 136.60, 132.50, 131.43, 128.32, 126.05, 125.36, 88.00, 83.75, 70.70, 61.63, 39.52 (overlap), 20.84, 19.71. HRMS (ESI): m/z [M+H]+ calcd for C19H22N5O4+ 384.1672, found 384.1662. 7-[N6-(2,4-dimethylbenzoyl)-5'-O-(4,4’dimethoxytrityl)- 2’-deoxyadenosine-3'-O-yl]-4,7-dioxoheptanoic acid, tri- ethylammonium salt (20): To a solution of 18 (42 g, 110 mmol) in pyridine (450 mL), DMTrCl (47 g, 140 mmol, 1.25 eq.) was added in five equal portions with an interval of 5 mins at 0 oC. After stirring for 2 hours at 0°C → r.t., MeOH (7 mL), NaHCO3 (20 g) and water (5 mL) were added suc- cessively to quench the reaction. After quenching, the mix- ture was concentrated to dryness. The residue was dis- solved in DCM (450 mL) and washed twice with water. The organic phase was separated, dried over Na2SO4, filtered and concentrated. The residue was purified by flash silica gel column chromatography (ethyl acetate/n-heptane, 1:2 → 2:1, v/v) to give 19 as white solid (37 g, yield: 50 %, HRMS (ESI): m/z [M+H]+ calcd for C40H40N5O6+ 686.2979, found 686.2990). To a solution of 4-oxoheptanedioic acid (20 g, 115 mmol, 2.0 eq) in anhydrous THF (300 mL), DMAP (2.8 g, 11 mmol, 0.2 eq.) and DCC (36 g, 170 mmol, 3.0 eq.) were added. The mixture was stirred for 16 hours at r.t. and fil- tered. A solution of 19 (37 g, 57 mmol) in THF (300 mL) and DBU (52 g, 340 mmol, 6 eq.) were added to the above fil- trate successively. After stirring overnight at r.t., the mix- ture was evaporated to dryness. The residue was purified by silica gel column chromatography (ethyl acetate/n-hep- tane, 1:1, v/v → DCM/MeOH/TEA, 100/2/1, v/v) to give 20 as white solid (38 g, yield: 74%). 1H NMR (500 MHz, DMSO-d6): δ 11.01 (s, 1H), 8.60 (s, 1H), 8.56 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.35 (d, J = 7.5 Hz, 2H), 7.23 (d, J = 8.6 Hz, 6H), 7.20 – 7.15 (m, 1H), 7.12 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.82 (t, J = 9.5 Hz, 4H), 6.51 (t, J = 7.0 Hz, 1H), 5.47 – 5.43 (m, 1H), 4.23 (s, 1H), 3.72 (s, 6H), 3.37 – 3.25 (m, 3H), 2.79 (t, J = 6.4 Hz, 2H), 2.70 – 2.61 (m, 5H), 2.55 (t, J = 6.4 Hz, 3H), 2.43 (s, 3H), 2.39 (t, J = 6.6 Hz, 2H), 2.32 (s, 3H), 1.00 (t, J = 7.2 Hz, 4.5H). 13C{1H} NMR (126 MHz, DMSO-d6): δ 207.8, 174.2, 171.9, 167.8, 158.1 (2 × C),6 151.8, 151.5, 150.2, 144.8, 143.4, 140.2, 136.7, 135.5, 135.5,9 132.5, 131.5, 129.7 (2 × C), 129.7 (2 × C), 128.4, 127.8 (2 × C), 127.7 (2 × C), 126.7, 126.1, 125.5, 113.1 13 (4C),32 85.7, 84.2, 83.5, 74.6, 63.8, 55.0 (2 × C), 45.3 (3 × C), 37.1, 36.6, 35.3, 28.6, 27.8, 20.9, 19.7, 10.3 (3 × C). HRMS (ESI): m/z [M-Et3NH++2H]+ calcd for C47H48N5O10+ 842.3401, found 842.3414. 7-[N6-(2,4-dimethylbenzoyl)-2'-deoxyadenosine-3'-O-yl] N-methyl-N-propargyl-4,7-dioxoheptane-amide (22): To a solution of 20 (38 g, 45 mmol) in DCM (400 mL), HOBT (9.1 g, 68 mmol, 1.5 eq.) and EDC (13 g, 68 mmol, 1.5 eq.) were added. After stirring for 2 hours at r.t., N-methyl-2- propynylamine (6.2 g, 90 mmol, 2.0 eq.) and Et3N (9.1 g, 90 mmol, 2.0 eq.) were added. The mixture was stirred for 12 hours at r.t. and washed twice with water. The organic phase was separated, dried over Na2SO4, filtered and evap- orated to dryness. The residue was purified by silica gel flash column chromatography (ethyl acetate/n-heptane, 1:1 →1:0, v/v) to give 21 as white solid (31 g yield: 96%, HRMS (ESI): m/z [M+H]+ calcd for C51H53N6O9+ 893.3874, found 893.3864). To a solution of 21 (30 g, 33 mmol) in DCM (300 mL), trifluoroacetic acid (5.7 g, 50 mmol, 1.5 eq.) was added. The mixture was cooled to 0 oC and 1-dodecanethiol (13 g, 66 mmol, 2.0 eq.) was added. The mixture was stirred at 0 oC for 2 h and washed twice with aqueous 5% NaHCO3. The organic phase was separated, dried over Na2SO4, fil- tered and evaporated to dryness. The residue was purified by preparative RP-HPLC (MeCN/H2O, 3:7 → 7:3, v/v) to give 22 as white solid (11 g, yield: 56%). 1H NMR (500 MHz, DMSO-d6): δ 11.03 (s, 1H), 8.71 (s, 2H), 7.48 (d, J = 7.8 Hz, 1H), 7.14 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.50 (dd, J = 8.4, 6.1 Hz, 1H), 5.40 (d, J = 5.8 Hz, 1H), 5.20 (t, J = 5.7 Hz, 1H), 4.19 (d, J = 2.2 Hz, 0.8H), 4.14 – 4.09 (m, 2.2H), 3.74 – 3.58 (m, 2H), 3.32 (t, J = 2.3 Hz, 0.4H), 3.15 (t, J = 2.4 Hz, 0.6H), 3.07 – 3.01 (m, 1H), 3.01 (s, 1.8H), 2.87 – 2.79 (m, 3.2H), 2.71 – 2.66 (m, 2H), 2.65 – 2.62 (m, 0.8H), 2.59 – 2.52 (m, 4.2H), 2.43 (s, 3H), 2.34 (s, 3H). 13C{1H} NMR (126 MHz, DMSO-d6) (note: rotamers observed): δ 208.0 & 208.0, 171.9, 171.1 & 170.9, 167.8, 151.9, 151.7, 150.2, 142.9, 140.2, 136.7, 132.5, 131.5, 128.4, 126.1, 125.3, 85.4, 83.9, 79.7 & 79.3, 75.1 & 74.9, 73.9, 61.6, 38.5, 36.8 & 36.5, 35.6, 33.9, 32.9, 27.9, 26.9 & 26.7, 20.7, 19.8. HRMS (ESI): m/z [M+H]+ calcd for C30H35N6O7+ 591.2567, found 591.2586. Tetrapodal soluble support 17: 22 (4.1 g, 6.9 mmol), tetrakis-O-(4-azidomethylphenyl)pentaerythritol15 (23, 0.9 g, 1.4 mmol) and sodium ascorbate (55 mg, 0.28 mmol, 0.2 eq) were dissolved in anhydrous dimethylacetamide (20 ml) and the solution was purged with argon for 60 min. CuI (0.16 g, 0.83 mmol) was added and the mixture was stirred for 48 h at r.t.. The solvent was removed under vacuum and the residue was partitioned between DCM and saturated NaHCO3. The organic phase was washed with 10 % EDTA disodium salt (3 x 20 ml), dried with Na2SO4 and evapo- rated to dryness. The crude material was purified by silica gel chromatography (MeOH/DCM, 5:10 → 10:9, v/v) afford- ing 17 (4.0 g, 96 %) as white solid. 1H NMR (500 MHz, CDCl3)(note: rotamers observed approximately in 0.3:0.7 ratio, cf. in Figures S28-S33): δ 8.72 (s, 4H), 8.38 (s, 1.2H), 8.37 (s, 2.8H), 7.56 (d, J = 7.9 Hz, 4H), 7.53-7.51 (m, 1.2H), 7.41 (s, 2.8H), 7.19-7.15 (m, 8H), 7.09 (s, 4H), 7.06 (d, J = 7.9 Hz, 4H), 6.90 – 6.83 (m, 8H), 6.45-6.38 (m, 4H), 5.52 (d, J = 5.5 Hz, 4H), 5.41 (s, 2.4H), 5.36 (s, 5.6H), 4.57 (s, 2.4H), 4.52 (s, 5.6H), 4.30 (s, 8H), 4.25 (m, 4H), 3.95 – 3.82 (m, 8H), 3.10-3.02 (m, 12.4H), 2.88 – 2.82 (m, 11.6H), 2.82-2.72 (m, 10H), 2.64 – 2.56 (m, 12H), 2.52 (s, 12H), 2.50-2.43 (m, 4H), 2.35 (s, 12H); 13C{1H} NMR (125 MHz, CDCl3): δ 208.1 & 207.9, 172.2 & 172.1, 171.6, 166.9, 159.1 & 159.0, 152.3, 150.7, 150.4, 144.4 & 144.2, 142.9 & 142.7, 142.0, 138.3, 132.7, 131.5, 129.8, 127.8, 127.4 & 127.2, 126.7, 124.1, 122.5 & 121.6, 115.3 & 115.2, 87.6 & 87.5, 87.2 & 87.2, 77.4 & 76.5, 66.6, 63.2, 53.9 & 53.7, 45.5 & 43.0, 44.8, 37.9 & 37.8, 37.3 & 37.3, 37.1, 35.5 & 33.9, 28.3 & 28.2, 27.3 & 27.2, 21.5, 20.5; MS (ESI + TOF): m/z calcd for [(M + 2H)/2]2+ 1511.6, found 1511.6. General procedure for one precipitation / synthesis cycle in LPOS: Coupling: To a solution of soluble support (0.165 mmol) in an anhydrous mixture of DMF (7 ml) and MeCN (4 ml), 5’-O-MIP protected phosphoramidite building block (1-4, 1.32 mmol) and 0.45 M tetrazole in MeCN (2.94 ml, 1.32 mmol) were added. The reaction was allowed to proceed for 2 hours at r.t., and quenched by addition of methanol (0.66 mmol). 2. Oxidation: m-chloroperbenzoic acid (77 m-%, 2.48 mmol, 15 eq) was added and the mixture was shaken for 5 min at r.t. 3. MIP-deprotection: dichloro- acetic acid (DCA, 2.47 ml, 15 % of total volume) was added and the mixture was shaken for 15 minutes at r.t. 4. Precip- itation: The reaction solution was added dropwise to 2-pro- panol (10 fold volume compared to the reaction mixture). The precipitated material was collected by centrifugation, washed with 2-propanol (100 ml) and dried under vacuum to afford soluble support-oligonucleotide construct (24, 25, 26, 28, 30 and 32) as white powder (cf. precipitation yields in Scheme 3). General procedure for two precipitations / synthesis cycle in LPOS: 1. Coupling: as above. 2. Oxidation: 0.2 M I2 in THF/Py/H2O (5:4:1 v/v/v) was added dropwise to the reac- tion mixture until the colour of iodine persisted and then the mixture was allowed to stir for further 15 minutes. The excess I2 was consumed by addition of trimethylphosphite until the colour of iodine cleared. 3. Precipitation: as above. 4. MIP-deprotection: The precipitate was dissolved in a mixture DCA-MeOH-DCM (5:18:27, v/v/v) and the mixture was stirred for 15 min, at r.t. 5. Precipitation: as above. The soluble support-oligonucleotide constructs (27, 29, 31 or 33) were obtained as white powders (cf. precipita- tion yields in Scheme 3). Aliquots of the precipitates were exposed to concen- trated ammonia (overnight at 55°C,) for evaluation of pu- rity, authenticity and yield (cf. Scheme 3) of the released di-, tri-, tetra- and pentanucleotide (Scheme S1). Solubility measurements. A saturation shake-flask method was adapted for probing the solubilities of the nu- cleotide linked soluble supports. In a sealed vial, an excess amount of the solid material was equilibrated with each tested solvent (500 µl) on an orbital shaker (400 rpm) at ambient temperature for 24 hours. An upper limit value for the solubility was set at 300 mg/ml and therefore, saturat- ing of samples reaching this value was not attempted. After the equilibration, the clear supernatant solution from each sample was transferred to a centrifuge tube, then briefly centrifuged, and an aliquot of the solution was diluted with dimethyl sulfoxide. The diluted samples were analysed with HPLC, and the mass concentrations of the analytes were determined by the UV peak area at 260 nm. The cal- culations were performed using a calibration curve fitted to the UV peak areas of five standard solutions of known mass concentrations prepared for each tested compound in dimethyl sulfoxide. Hydrazine acetate cleavage of protected trinucleotides (36 and 37). 5’-O-MIP-protected trinucleotide-soluble sup- ports (34 and 35) were assembled on 40 and 90 µmol scale by following the LPOS procedures above. Overall precipi- tation yields of 96 and 92%, respectively, were obtained. A solution of 0.1 M hydrazine acetate was prepared by adding hydrazine hydrate (25 µl) to a mixture of acetic acid (1 ml) and pyridine (5 ml). Trinucleotide-soluble support con- struct 34 (190 mg, 29 µmol) was dissolved in the freshly prepared 0.1 M hydrazine acetate (1.4 ml, 140 µmol, 5 eq) and the mixture was stirred for 1 hour. The reaction solu- tion was diluted with DCM and the organic phase was washed with saturated NaHCO3. The organic phase was dried with Na2SO4 and concentrated under vacuum. The crude material was purified by a short silica gel column (MeOH/DCM, 5:95 → 7:93, v/v, containing 1 % pyridine) affording 36 as white solid (106 mg, 71 %). Trinucleotide- soluble support construct 35 (575 mg, 85 µmol) was treated in a similar manner to afford 36 (292mg, 65 % yield) as white powder. See 1H NMR (500 MHz, CD3CN), 13C{1H} NMR (126 MHz, CD3CN) and 31P NMR (202 MHz, CD3CN) spectra of 36 and 37 in Figures S34-S43. 36: MS (ESI-TOF): m/z calcd for [M + H]+ 1287.4, found 1287.4. 37: MS (ESI- TOF): m/z calcd for [M + H]+ 1318.5, found 1318.5. Funding Sources PV and PR acknowledge ACS Green Chemistry Institute Pharmaceutical Roundtable research grant 2020: https://www.acsgcipr.org/advancing-research/ ASSOCIATED CONTENT Data Availability Statement The data underlying this study are available in the published article and its Supporting Information. AUTHOR INFORMATION Corresponding Author * Pasi Virta – Department of Chemistry, University of Turku, 20500 Turku, Finland; https://orcid.org/0000-0002-6218- 2212; Email: pamavi@utu.fi Author Contributions The manuscript was written through contributions of all au- thors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally. (match statement to author names with a symbol) Notes The authors declare no competing final interest. Supporting Information NMR data for new compounds (2-6, 9, 12, 15, 17, 18, 20, 22, 36 and 37), RP HPLC profiles, including purity according to peak areas of the HPLC profiles, and MS spectra of di-, tri-, tetra- and pentanucleotides released from oligonucleotide-soluble support constructs 24-33. MS spectra of oligonucleotide-solu- ble support constructs 32 and 33. Graphic illustrations of mass content and varying solubility of oligonucleotide-soluble sup- port constructs. HPLC monitoring of hydrazine acetate-medi- ated orthogonal cleavage of a protected trinucleotide. The Supporting Information is available free of charge on the ACS Publications website. ACKNOWLEDGMENT The authors would like to thank Dr. Maarit Karonen for MS (ESI-TOF) analysis of the oligonucleotide-soluble sup- port constructs. This manuscript was developed with the support of the ACS Green Chemistry Institute Pharmaceutical Roundtable (www.acsgcipr.org). 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