Solid-Phase Synthesis of Oligonucleotide Conjugates Useful for

JUNE 2009 Volume 20, Number 6  Copyright 2009 by the American Chemical Society

REVIEWS Solid-Phase Synthesis of Oligonucleotide Conjugates Useful for Delivery and Targeting of Potential Nucleic Acid Therapeutics Harri Lo¨nnberg* Department of Chemistry, University of Turku, FIN-20014 Turku, Finland. Received September 23, 2008; Revised Manuscript Received November 13, 2008

Olignucleotide-based drugs show promise as a novel form of chemotherapy. Among the hurdles that have to be overcome on the way of applicable nucleic acid therapeutics, inefficient cellular uptake and subsequent release from endosomes to cytoplasm appear to be the most severe ones. Covalent conjugation of oligonucleotides to molecules that expectedly facilitate the internalization, targets the conjugate to a specific cell-type or improves the parmacokinetics offers a possible way to combat against these shortcomings. Since workable chemistry is a prerequisite for biological studies, development of efficient and reproducible methods for preparation of various types of oligonucleotide conjugates has become a subject of considerable importance. The present review summarizes the advances made in the solid-supported synthesis of oligonucleotide conjugates aimed at facilitating the delivery and targeting of nucleic acid drugs.

INTRODUCTION Selective inhibition of gene expression by oligonucleotides, including single-stranded antisense oligonucleotides (1-8), ribozymes (9), and double-stranded siRNAs (10-12), shows promise as a novel form of chemotherapy, which instead of proteins uses nucleic acids as targets. In addition, aptamers (13) and immunostimulatory CpG-oligonucleotides (14) exhibit potential as therapeutic agents recognizing proteins. While some of these approaches, above all antisense and siRNA technology, are extensively used for target validation of conventional small molecule drugs, the pathway to exploitation of oligonucleotides themselves or their structural analogues as drugs has been long; despite considerable advances, several thresholds still remain to be overcome (15-21). Among the existing problems, inefficient cellular uptake and subsequent release from endosomes to cytoplasm appear to be the most severe ones. In addition, cell type or organ specific targeting, proper intracellular distribution, and * Corresponding author. Phone +358-2-333 6770, Fax +358-2-333 6776, E-mail: [email protected].

increased plasma half-lives are subjects that play a central role in development of oligonucleotide-based therapeutics. 1

ABBREVIATIONS: Ac, Acetyl; All, Allyl; Alloc. Allyloxycarbonyl; Bhoc, Benzhydryloxycarbonyl; Bn, Benzyl; Boc, tert-Butoxycarbonyl; Bpoc, 2-(Biphen-4-yl)propan-2-yloxycarbonyl; Bz, Benzoyl; 2-ClTr, 2-Chlorotrityl; CPG, Controlled pore glass; DBU, 1,8-Diazabicyclo[5.4.0]undec7-ene; DCM, Dichloromethane; DIPEA, N,N-Diisopropylethylamine; DMTr, 4,4′-Dimethoxytrityl; Dmab, 4-{N-[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}-benzyl; Dnp, 2,4-Dinitrophenyl; Fm, 9-Fluorenylmethyl; Fmoc, 9-Fluorenylmethoxycarbonyl; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexaflorophosphate N-oxide; HOBt, 1-Hydroxybenzotriazole; iBu, Isobutyryl; LDL, Low density lipoprotein; Lev, Levulinoyl; MBHA, Methylbenzhydrylamine; MMTr, 4-Methoxytrityl; MSNT, 1-(2-Mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazolide; ODN, Oligodeoxyribonucleotide; ORN, Oligoribonucleotide; PEG, Poly(ethylene glycol); PhiPr, Phenylisopropyl (1-methyl-1-phenylethyl); Pix, 9-Phenylxanthen-9-yl; PNA, Peptide nucleic acid; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; RISC, RNA-induced silencing complex; TBDMS, tert-Butyldimethylsilyl; TBDPS, tert-Butyldiphenylsilyl; TBTU, O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TCA, Trichloroacetic acid; TFA, Trifluoroacetic acid; Tfa, Trifluoroacetyl; THF, Tetrahydrofuran; Tol, Toluoyl; Tr, trityl.

10.1021/bc800406a CCC: $40.75  2009 American Chemical Society Published on Web 01/28/2009

1066 Bioconjugate Chem., Vol. 20, No. 6, 2009 Scheme 1

Chart 1

Lo¨nnberg

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Bioconjugate Chem., Vol. 20, No. 6, 2009 1067

Chart 2

Scheme 2

Conjugation of oligonucleotides with molecules that provide the conjugate with a desired novel property offers a feasible way to respond to these requirements. For example, oligonucleotides bearing a conjugate group known to be internalized by receptor-mediated active transport expectedly exhibit

enhanced uptake compared to their nonconjugated counterparts (22-24). Cell-penetrating peptides, glycoclusters, lipids, polyamines, and numerous small molecules have all received interest as possible carriers of oligonucleotides. Although none of these approaches has so far led to a real breakthrough,

1068 Bioconjugate Chem., Vol. 20, No. 6, 2009

Lo¨nnberg

Chart 3

Scheme 3

many encouraging observations have been made. Conjugation of neutral peptide nucleic acid (PNA1) or phosphoramidate morpholino analogues of oligonucleotides to arginine-rich peptides has been reported to significantly enhance their cellular uptake and strengthen their antisense effect (25-29).

Galactosylated conjugates of oligodeoxyribonucleotide (ODN) phosphorothioates are internalized via the galactose-specific asialoglycoprotein receptor-mediated endycytosis (30). Poly(ethylene glycol) (PEG) conjugation has been shown to prolong the half-life in plasma (31), and conjugation with

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Scheme 4

Chart 4

lipophilic groups (32-34) or aptamers (35) has increased the efficiency of siRNA-induced gene silencing in vivo. Owing to these and many other promising results, development of efficient and reproducible methods for convenient preparation of various types of oligonucleotide conjugates has become a subject of considerable importance. Workable chemistry is a prerequisite for extensive biological studies. The methods used for the preparation of oligonucleotide conjugates fall in two major categories: solution- and solidphase conjugation. On applying the solution-phase approach,

the oligonucleotide is at some stage of its solid-phase synthesis equipped with an appropriate functionality, to which the conjugate group is coupled in solution after release and purification of the oligonucleotide. When the solid-phase methodology is used, the entire conjugate is assembled on a single support, either by conjugation of a prefabricated conjugate group or by assembling the conjugate moiety by a stepwise process prior to or after the oligonucleotide synthesis. The advantage of the solid-supported method compared to conjugation in solution is a less laborious


Lo¨nnberg

Chart 5

purification. To achieve high coupling efficiency, the conjugate group is usually used in considerable excess, which complicates the purification when the conjugation is carried out in solution. By contrast, on a solid support, the unreacted conjugate group and the possible side products may be removed by simple washing, which markedly facilitates the chromatographic purification after release from the support. The use of a solid support may also help to avoid the problems otherwise arising from limited solubility of one of the reactants. The present paper reviews the methods described for the solidsupported synthesis of oligonucleotide conjugates useful for delivery and targeting of potential nucleic acid therapeutics. Accordingly, preparation of oligonucleotides conjugated with lipids, polyamines, peptides, carbohydrates, PEG, and some small molecules are discussed. In addition, synthetic procedures for oligonucleotides bearing biodegradable or thermolabile phosphate protecting groups are summarized.

HYDROPHOBIC CONJUGATES Since the hydrophobic interior of the phospholipid bilayer forms the main obstacle to internalization of polyanionic oligonucleotides, it is quite natural that conversion of oligonucleotides to their lipid conjugates has long received interest as a possible method to enhance their cellular uptake (36). Reduction of the hydrophilic nature of the oligonucleotide is not, however, the only reason for lipid conjugation. Some hydrophobic pendants, above all cholesterol, are believed to bind to lipoproteins and particles of low-density lipoproteins (LDL), a cholesterol transporter in blood. Consequently, the cholesterol conjugate becomes recognized by hepatitic cells (37-39) and, hence, internalized by lipoprotein-mediated endocytosis. As mentioned above, conjugation with cholesterol has been shown to increase the efficacy of siRNA-induced silencing of the apoB mRNA gene in rodents’ liver and jejunum (32). The hydrophobic conjugates of oligonucleotides are usually obtained by one of the following solid-phase approaches: (i) 3′-conjugates by chain assembly on a support bearing the desired

conjugate group, (ii) 5′-conjugates by introduction of the conjugate group as a phosphoramidite reagent, and (iii) intrachain conjugates by making use of prefabricated nucleosidic building blocks or their congeners incorporating the conjugate group. Scheme 1 shows an illustrative example of the preparation of the 3′-conjugates. A branched alcohol bearing orthogonally protected amino and hydroxyl groups is attached to the support via a succinyl linker. The conjugate group bearing a hydroxyl function is first attached to the deprotected amino group of this handle as a chloroformate ester, and the ODN is then assembled on the deprotected hydroxyl function (40). Support 1 (Chart 1) had earlier been used similarly (41). While in these cases a stepwise immobilization has been applied to obtain the cholesterol bearing support, related supports have more often been prepared by immobilization of a prefabricated linker-cholesterol conjugate (34, 42-48). Among these, support 6 has been used for construction of oligoribonucleotide (ORN) conjugates (34) and 2-5 for the synthesis of ODN conjugates. Supports 4 derived from trans-L-hydroxyprolinol have additionally been used for the preparation of ODN conjugates of many other steroids and diacylated glycerols (49). 2′-O- (50) and 3′O-succinylated uridine (51), 7 and 8, conjugated with cholesterol via the remaining secondary hydroxyl function represent still another type of support utilized for preparation of the 3′-terminal conjugates. Chart 2 shows the structures of the phosphoramidite (9-15) and H-phosphonate (16) reagents, both non-nucleosidic and nucleosidic ones, used to introduce hydrophobic conjugate groups into the 5′-terminus of ODNs (46, 47, 49, 51-55). Building block 13 deserves special attention. This phosphoramidite reagent derived from the allyl ester of 3-R-(2-hydroxyethoxy)cholic acid is obtained by selective phosphitylation of the primary hydroxyl group with 1.1 equiv of 2-cyanoethylN,N-diisopropylphosphorochloridite without interference of the secondary hydroxyl functions (54). The allyl protection may then be removed on-support with Pd[PPh3]4. 5′-Palmitoyl ODNs have been obtained by using base-labile t-butylphenoxyacetyl protecting groups for base moiety amino functions and oxalyl

Reviews


Scheme 5

Chart 6

linker. These modifications to the standard phosphoramidite strategy allow mild deprotection with ethanolamine, and hence, the 5′-ester linkage remains intact (56). Incorporation of hydrophobic groups into intrachain positions of the oligonucleotide chain is usually performed with the aid of prefabricated nucleosidic or non-nucleosidic building blocks. The nucleosidic building blocks exploited include the phosphoramidites of C5-derivatized pyrimidine 2′-deoxyribonucleosides (17, 18) (57, 58), 2′-O-derivatized uridine (19, 20) (50, 51), and a thymine amino-LNA monomer (21) (59). Several hydrophobic conjugate groups including cholesterol (50, 57, 59), R-tocopherol (58), adamantanylacetic acid (50), long chain fatty acids (50), and diacylglycerol (50) have been introduced in this manner (Chart 3). A prolonged coupling time is required to couple the lipidated block (57, 58). Octyl groups have been incorporated into ODNs using a phosphoramidite building block derived from dioctyl 2,2-bis(hydroxymethyl)malonate (60). Again, a prolonged coupling time (10 min) is required for high coupling yield (>98%) and the subsequent detritylation has to be carried out with TFA. Interestingly, the ester linkages at internucleosidic positions withstand normal ammonolysis. Besides exploitation of prefabricated solid supports and phosphoramidite reagents bearing the conjugate group, several on-support conjugation procedures have been reported. In fact, the first solid-phase synthesis of a cholesterol conjugate is a nice example of such an approach, involving oxidative phosphoramidation of a support bound dinucleoside-3′,5′-(H-phos-

phonate) with CCl4 in the presence of cholester-3-yloxycarbonylaminoethylamine and subsequent assembly of the ODN chain (Scheme 2) (61). Interestingly, one of the very recent approaches follows a related principle. Instead of conventional succinyl linker, an aminoalkyl support is acylated with 3-chloro-4hydroxyphenylacetic acid, and the 3′-terminal nucleoside is coupled to the hydroxyl function by the normal phosphoramidite chemistry (Scheme 3) (62). The 2-cyanoethyl protection is then removed with triethylamine in pyridine (1 h, 50 °C) and the conjugate group, e.g., cholesterol, is coupled to the exposed phosphodiester linkage by the phosphotriester chemistry. On using MSNT as an activator, the coupling yield has been reported to range from 75% to 80%. The ODN chain is then assembled by phosphoramidite chemistry. Upon ammonolysis, only the ODN bearing the conjugate group is released from the support, since the phosphodiester linkage is not cleaved by ammonolysis, not even when aryl pohosphodiesters are concerned. It is also possible to remove the 2-cyanoethyl protection and assemble the ODN chain prior to the introduction of the conjugate group. In case one wishes to conjugate amines instead of alcohols, the phosphodiester linkage is first tosylated, and a nucleophilic displacement with the amine is then carried out. Thiocholesterol has been conjugated to the 5′-terminus of a support-bound ODN by elongation of the chain with 6-chloroacetamidohexanol phosphoramidite and displacing the chloro substituent with thiocholesterol in dioxane in the presence of DBU. Normal ammonolysis then releases the conjugate (Scheme 4A) (63). Long chain aliphatic alkyl groups and small aromatic groups have been introduced similarly. Several lipophilic alcohols, including cholesterol, borneol, menthol, and heptadecanol, have been converted to 2-alkoxy-2-thiono-1,3,2-oxathiaphospholane and reacted in DCM in the presence of DBU with


Lo¨nnberg

Chart 7

the 5′-hydroxy group of a support-bound ODN (Scheme 4B) (64). Again, a normal ammonolysis is finally carried out. Masking of the internucleosidic phosphodiester bonds of an antisense oligonucleotide with biodegradable or thermolabile protecting groups represent another type of utilization of hydrophobic conjugate groups. The most extensively studied examples are S-acyl-2-mercaptoethyl and alkoxymethyl-protected ODNs. The former pro-oligonucleotides have been assembled by the phosphoramidite chemistry using S-tert-butyl2-mercaptoethyl-N,N-diisopropylphosphoramidites instead of the 2-cyanoethyl protected ones (65). Since the phosphotriester linkages do not withstand ammonolysis, the base moiety acyl protecting groups have been replaced with photolabile 6-nitroveratryloxycarbonyl or 2,2′-bis(2-nitrophenyl)ethoxycarbonyl group, and the synthesis has been carried out on a photolabile 1-(2-nitrophenyl)-1,3-propanediol linker. For the S-acetyl-2mercaptoethyl protected oligomers, three alternative strategies have been proposed. First, the chain assembly has been carried out on a photolabile linker, but the base moieties have been protected with a Pd(0)-labile allyloxycarbonyl group and those phosphodiester linkages that do not bear a 2-methylthioethyl group with an allyl group (66). Second, fluoride-ion-labile

protections, viz., 4-(tert-butyldiphenylsilyloxymethyl)benzoyl group for the base moieties and 2-(trimethylsilyl)ethyl group for the phosphate moieties, have been used (67). When the oligomer is assembled on a phosphoramidate linker, it is released by the Et3N · (HF)3 treatment used to remove the base and phosphate protections. The third alternative is chain assembly on a disulfide linker using building blocks that bear 2-(trimethylsilyl)ethoxycarbonyl protections on nucleobases and 2-(trimethylsilyl)ethyl protection on phosphate moieties (68). The base moiety and phosphate protections are removed by a single ZnBr2-treatment on the support and the deprotected oligomer is released reductively into the solution. O-(4Acyloxybenzyl)phosphorothioate linkages have also been sitespecifically introduced into oligonucleotides by the phosphoramidite strategy using building blocks bearing a 4-acyloxybenzyl group instead of the 2-cyanoethyl group. N-Pent-4-enoyl group, cleaved upon release of the oligomer from the support with methanolic K2CO3, has been used for the base moiety protection (69). 3-Pivaloyloxy-2,2-bis(ethoxycarbonyl)propyl protections have been introduced into ODNs as prefabricated dimeric phosphoramidite reagents (70).

Reviews Scheme 6

All the pro-oligonucleotides discussed above bear esteraselabile protecting groups. In addition, oligonucleotides bearing spontaneously cleavable phosphate protections have been suggested to be viable antisense prooligonucleotides. For this purpose, ODNs bearing 3-hydroxypropyl (71), 4-hydroxybutyl (71), or N-formyl-N-methyl-2-aminoethyl (72) protecting groups on selected internucleosidic phosphodiester linkages have been prepared. The departure of all these groups is based on thermolytic cyclization. For the synthesis, conventional phosphoramidite chemistry may be applied when nucleoside 3′-(N,Ndiisopropylphosphoramidites) bearing the desired protecting groups are used and normal ammonolysis is replaced by treatment with pressurized ammonia at 25 °C.

POLYAMINE CONJUGATES Polyamines, being positively charged at physiological pH, form complexes with negatively charged oligonucleotides. These complexes, known as polyplexes, interact with the negatively charged phospholipids of the cell membrane and, hence, enhance the cellular uptake (73). Dendrimeric polyamines, for example, are extensively used as transfection agents for plasmids, but also for siRNA and oligonucleotides. Accordingly, it is no surprise that covalent polyamine-oligonucleotide conjugates have received attention as potential facilitators of the delivery of therapeutic oligonucleotides. In addition, it is worth noting that some oligoamines, such as spermine and spermidine, are naturally occurring metabolites known to complex in cells with RNA (74), and they may, hence, affect the biodistribution of their oligonucleotide conjugates. Oligoamines, either linear or branched, are usually attached to the 5′-terminal hydroxyl function of the chain, to a C5 site

Bioconjugate Chem., Vol. 20, No. 6, 2009 1073 Scheme 7

of a pyrimidine base or to 2′-O of a ribonucleoside unit. Up to 6 R,ω-bis(4-hydroxybutyl)spermine molecules have been incorporated as trifluoroacetyl protected phosphoramidite reagents (23; Chart 4) into the 5′-terminus of ODNs, giving a long linear spermine-phosphate tail (75). The coupling efficiency of these building blocks has been reported to be as high as 90-96%. Standard ammonolysis is adequate to remove all trifluoroacetyl protections. A single C-branched spermine has been incorporated as a phenoxyacetyl protected phosphoramidite reagent (24) (76, 77). More complex 5′-terminal polyamine structures have been constructed by coupling a 5′-amino-5′-deoxythymidine phosphoramidite bearing a spruce-like methoxyoxalamido-functionalized polyamine (25) as the last building block of the chain assembly. Upon release and deprotection of the conjugate with a primary amine, e.g., spermidine or histidine, each branch becomes further derivatized with this amine by formation of oxalyldiamide linkages (78). Tetrazole activation with 15 min reaction time has been used for coupling of this highly branched block. Several spermine moieties have been incorporated into ODNs with the aid of appropriately protected phosphoramidites of N2derivatized 2′-deoxyguanosine (26; Chart 5) (79, 80), N6-


Lo¨nnberg

Chart 8

derivatized 2′-deoxyadenosine (27) (80, 81), and N4-derivatized 2′-deoxycytidine (28a) (82) and its 5-methyl analogue (28b) (83). The cytidine derivative coupled as well as the unmodified 2′-deoxynucleoside building blocks, but for the guanosine derivative, repeated coupling and prolonged coupling time (10 min) had to be applied to obtain a coupling efficiency of 95%. Into ORNs, spermine moieties have been incorporated with a C5-modified 5′-O-DMTr-2′-O-TBDMS-uridine 3′-phosphoramidite building block (29) (84). A coupling efficiency of 96% has been achieved by using 5-benzylthiotetrazole as an activator. Besides the phosphoramidite chemistry, oligoamines have been introduced into oligonucleotides by on-support nucleophilic displacement and oxidative amidation. 5-Methoxycarbonylmethyl-2′-deoxyuridine (85) or 2-fluoro-2′-deoxyinosine (86) has been incorporated into a desired site of the sequence, and treatment with sperimine has then resulted in conjugation by aminolysis of the methoxycarbonyl group upon release and deprotection of the conjugate. Similarly, treatment of the electrophilic conjugate depicted in Scheme 4A with spermine gives the desired oligoamine conjugate (63). Spermine can be used as a cleaving agent in an unprotected form, since the primary amino group is a much more efficient nucleophile than the secondary one. A branched oligoamine, N1,N1-bis(2-aminoethyl)ethane-1,2-diamine, has, in turn, been conjugated by oxidative amidation (87). For this purpose, the 5′-hydroxy function has been phosphitylated with (anthracen-9-yl)methyl 2-cyanoethyl N,N-diisopropylphosphoramidite and oxidation with I2 in THF in the presence of the appropriately protected amine has then been carried out (Scheme 5). 98% of the (anthracen-9-yl)methyl group becomes oxidatively displaced with the amine. A special feature of this kind of conjugation is that the phosphoramidate linkage is acid-labile and may, hence, be cleaved, although expectedly quite slowly, at the relatively low pH of endosomes (pH 5). Another possible way to introduce several aliphatic amino groups into an oligonucleotide is incorporation of several modified nucleosidic building blocks, each bearing a single amino group, into the chain along its entire length. 2′-O has

been regarded as an appropriate site for tethering of aminoalkyl groups, since 2′-O-alkylated oligonucleotides hybrizide even better than their 2′-deoxy counterparts (88). In addition, the catalytic argounaut proteins within the RISC complex appear to tolerate well 2′-O-modifications of siRNA (89). All canonical ribonucleosides have been prepared as 2′-O-(N-phthaloyl-3aminopropyl) derivatives and introduced into oligonucleotides as appropriately protected phosporamidites (90). The oligonucleotides prepared contained long, contiguous sequences of the aminoalkylated nucleosides. A coupling efficiency of 98.5% has been achieved by repeated coupling steps. In addition to normal ammonolysis, a treatment with 10% aq methylamine has been applied to remove the phthaloyl protections. Several contiguous 2′-O-(3-dimethylaminopropyl)- (91) and 2′-O-[2(2-dimethylaminoethoxy)ethyl]-5-methyluridine (92) blocks have been introduced without any amino protection, whereas 2′-O(6-aminohexyl)-adenosine and -uridine blocks have been inserted as trifluorocetamides (93). The shortcoming of the attachment of bulky groups to 2′-O, however, is reduced coupling efficiency. For example, the phosphoramidite 30 (Chart 6) bearing a bulky Boc-protected carboxyspermine has been reported to couple only in 40% yield (88). By contrast, phosphoramidite 31, having an ara configuration and bearing a branched phenoxyacetyl protected spermine attached to the 2′-O via a phosphotriester linkage, has been observed to couple without marked difficulty (76, 77). Oligonucleotides bearing up to five contiguous 2′-O-(N-lysyl-6aminohexyl)uridines (32), introduced in N-trifluoroacetylated form, have been successfully assembled on a solid support by using extended coupling times (94, 95). Like amino groups, guanidinium groups remain protonated and, hence, positively charged at physiological pH. ODNs bearing 2′-O-[2-(guanidinium)ethyl] groups have been prepared by using phosphoramidite 33 (Chart 7), where the guanidinium moiety is protected with 2-cyanoethoxycarbonyl groups (96). tert-Butylhydroperoxide or 1-S-(+)-(10-camphorsulfonyl)oxaziridine has been used for the oxidation and the 2-cyanoethoxycarbonyl groups have been removed with piperidine before normal ammonolysis to

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Scheme 8

prevent cyclization of the guanidinium group to triazine. Aminoalkyl groups have also been tethered to C4′ of thymidine, and several such modified nucleosides (34, 35) have been incorporated into ODNs by the phosphoramidite chemistry (97, 98). A 96% coupling efficiency has been achieved on using a prolonged coupling time (6 min) and a high phosphoramidite concentration (1.2 mol L-1). Besides the sugar hydroxyl functions, C5 of pyrimidine nucleosides has been used as a site for the attachment of aminoalkyl groups. Several 2′-deoxyuridines and 2′-deoxycytidines bearing linear (99-102) or branched (103, 104) aminoalkyl side chains at C5 have been incorporated into ODNs by normal phosphoramidite chemistry. Immobilization of the corresponding nucleosides to an aminoalkylated solid support via a succinyl linker has allowed synthesis of conjugates having the 3′-terminal nucleoside modified also (100, 103, 105). Additionally, a postsynthetic approach has been applied to obtain aminoalkyl-derivatized oligonucleotides. 5-(Methoxycarbonyl)2′-deoxyuridine has first been introduced into the ODN chain, and treatment with ethane-1,2-diamine or hexane-1,6-diamine has then resulted in aminolysis of the methoxycarbonyl function

to an ω-aminoalkylcarbamoyl function upon the release and deprotection of the conjugate (106). ODNs bearing an aminoalkylated phosphoramidate tail at the 3′-terminus have been prepared by assembling first a nonnucleosidic backbone for the tail from 3-DMTrO-1-methylpropyl H-phosphonates and oxidizing the H-phosphonate diester linkages obtained with CCl4 in the presence of N-trifluoroacetylbutane-1,4-diamine (Scheme 6). The oligonucleotide chain is then assembled by phosphoramidite chemistry (107). Similarly, ODNs bearing a 5′-polyimidazole tail have been prepared by consecutive coupling of several 4-DMTrO-butyl H-phosphonates to the 5′-terminus of an otherwise fully protected support-bound ODN and subsequent oxidation with CCl4 in the presence of histamine (108).

PEPTIDE CONJUGATES Some rather short peptides have been discovered to translocate across cell membranes, partly by endocytic processes but partly by endocytosis-independent cytoplasmic entry (109). Such peptides usually are Arg- or Lys-rich. The two naturally

1076 Bioconjugate Chem., Vol. 20, No. 6, 2009 Scheme 9

Lo¨nnberg Scheme 11

Scheme 10

occurring sequences, Penetratin and Tat peptide, and a synthetic hybrid, Transportan, of two naturally occurring sequences are

the best-known examples (110). Conjugation of antisense oligonucleotides with these cell-penetrating peptides has received wide interest as a possible way to enhance their delivery into cells (111-113). In addition, conjugation with nuclear localization signal peptides has been studied as a means to affect the intracellular distribution of oligonucleotides (114). It is not, hence, surprising that synthesis of peptide conjugates has been the subject of more intensive studies than any other category of oligonucleotide conjugates. The progress of the field has been reviewed every few years (115-119). In the present review, attention is only paid to alternative approaches of solid-supported synthesis and, in particular, to novel approaches applicable to preparation of reasonably long peptide conjugates. The solutionphase conjugation of prefabricated peptides and oligonucleotides bearing mutually reactive functionalities is not discussed, although such methods are often highly useful and still more extensively used than solid-phase syntheses (116-118, 120). Although the solid-phase synthesis of both oligonucleotides and peptides is well-established, assembly of oligonucleotidepeptide conjugates from monomeric units on a single support still is a considerable challenge. The existing protocols of oligonucleotide synthesis are largely based on acid-labile temporary protection and base-labile global protection, whereas the opposite is true for the peptide synthesis, or alternatively, both the temporary and global protecting groups are acid-labile. Accordingly, at least one of the existing strategies must be considerably modified to enable a stepwise chain assembly. The stepwise strategies suggested so far fall into two major categories: either a branched linker bearing an amino group for peptide synthesis and a hydroxyl function for subsequent

Reviews


Scheme 12

Scheme 13

assembly of the oligonucleotide sequence is utilized, or a linker bearing a hydroxyl function is attached to the amino terminus of the support-bound peptide and used as a starting point for the oligonucleotide chain assembly. Both approaches face the same difficulty, viz., incompatibility of the protecting group strategies usually applied to oligonucleotide and peptide synthesis. As far as the peptide moiety containing no side-chain fuctionalities, or only those that can be protected with orthogonally removable groups or such base-labile groups that withstand the Fmoc removal with piperidine, the situation is not very problematic. The synthesis may be carried out on a branched linker bearing an Fmoc-protected amino group and a DMTrprotected hydroxy function. The peptide moiety is first assembled on the amino group by the Fmoc chemistry and the ODN moiety then on the hydroxyl function by the phosphoramidite chemistry. The early examples of such an approach are offered by the synthesis of a conjugate of 17-mer ODN and a 3′-terminal tripeptide on linker 37 (Chart 8) (121) and a conjugate of a 23-mer ODN and a base moiety bound dipeptide on linker 38 (122). Among these, 37 may be cleaved by successive treatments with ethane-1,2-diamine in ethanol and IO4- in aqueous acetonitrile and 38 by normal ammonolysis. More recently, a branched linker strategy (39) has been applied to preparation of a 14-mer phosphorothioate ODN bearing a 16-mer membrane permeable peptide, viz., the hydrophobic region of the signal peptide sequence of the Kaposi fibroblast growth factor (123). While this sequence does not contain any trifunctional amino acids, incorporation of some such residues appears feasible. Linker 40 has been successfully used for

synthesis of peptide conjugates containing Cys, Lys, and Ser residues (124). Their side-chain functionalities have been protected with Tr, Boc, and TBDMS groups, respectively. Although these protecting groups are acid-labile, they have been reported to tolerate the repeated removal of DMTr protections during the assembly of a 15-mer ODN. They have been removed after the ODN assembly, but before the ammonolysis. When an even more generally applicable procedure is required, exchange of the acid-labile side-chain protecting groups of the peptide moiety to base-labile acetyl protections before assembly of the ODN chain appears to be an attractive alternative (125). The synthesis is carried out on linker 41, the essential part of which is NR-Fmoc-Nε-Boc-lysine anchored to the support through an ester linkage (Scheme 7). The peptide moiety is assembled by the normal Fmoc chemistry on the R-amino group of this lysine handle. The acid-labile side-chain protections, including Tr on histidine, Boc on tryptophan, and tBu on serine, are removed, as well as the Boc protection of the lysine handle. The exposed ε-amino group is acylated with DMTr-protected glycolic acid, and the side-chain functionalities of the peptide moiety are acetylated. The oligonucleotide chain is then assembled on the detritylated hydroxy function of the glycolic acid residue by the phosphoramidite strategy. The conjugate obtained is deprotected and cleaved from the support with aqueous sodium hydroxide. Apart from arginine, this protocol allows introduction of all trifunctional amino acids. Arginine residues have to be incorporated in the sequence as ornithines and converted postsynthetically to arginines by treatment with N,N′-bis-Fmoc-guanidine-N′′-triflate (126). Peptide conjugates of ORNs have been prepared by using an aminonucleoside as a branched linker (Scheme 8). An appropriately protected 3′-amino-3′-deoxyadenosine bearing a Fmoc protected alanyl group at 3′-N is immobilized to an aminofunctionalized support via a 2′-O-succinyl linker (127). A peptide chain, containing only hydrophobic or allyl ester protected amino acids, is assembled on the alanyl residue by the Fmoc chemistry, after which the ORN sequence is assembled


Lo¨nnberg

Scheme 14

Scheme 15

on the 5′-OH by the TBDMS/phosphoramidite chemistry. The allyl protections are removed with Pd(PPh3)4 on the support and the silyl protections in solution after ammonolysis. Another example of the utilization of an aminonucleoside as a branched linker is synthesis of ODN phosphorothioates bearing a 2′-Otrilysylsuccinyl tail on the 3′-terminal nucleoside. 2′-Deoxy-2′(2-benzyloxy-1,2-dioxoethylamino)uridine has been immobilized to a PEG-grafted support via a 3′-succinyl linker (128). After the ODN chain assembly, the benzyl group is reductively removed and N-trifluoroacetyllysine benzyl ester is coupled to the exposed carboxy function using carbodiimide activation. The procedure is repeated until the desired number of Lys residues have been coupled. Ammonolysis completes the synthesis. Still, one possible way to use a nucleoside as a branched linker is to immobilize it through the base moiety to the support. 3′-O-TBDMS-5′-O-DMTr-2′-deoxycytidine has been immobilized through the N4-atom via a succinyl linker to an aminoalkylated support (129). The ODN moiety is then assembled on the detritylated 5′-OH in an inverse direction using appropriately protected nucleoside 5′-phosphoramidites. After desilylation, the 3′-hydroxy group is phosphitylated with N-MMTr-6-aminohexylphosphoramidite, and the deprotected amino group is used as a starting point for the synthesis of the peptide moiety by the Fmoc chemistry. Instead of a branched linker, the hydroxyl function serving as the starting point of the ODN synthesis may be provided by an N-terminal ω-hydroxyacyl group or by a side-chain hydroxyl function of an N-terminal amino acid. The limitations concerning

the side-chain protections of the peptide moiety are naturally similar to those using a branched linker. On applying this methodology, the peptide synthesis has often been carried out by the Boc chemistry on a base-labile 2-nitrophenylethanol (42) or 9-fluorenylmethanol linker (43) and the ODN chain is then assembled on a side-chain hydroxyl functionality of the Nterminal amino acid, viz., tyrosine, threonine, or serine (Scheme 9) (130-137). Use of homoserine instead of serine appears, however, preferable, since this increases the stability of the conjugate toward basic conditions (138, 139). When desired, the ODN synthesis may also be initiated from a nonterminal amino acid, usually serine or homoserine (138, 140). The side chains are protected with base-labile groups: Asp with Fm (132, 133), His with Dnp (138), Lys with Fmoc or Tfa (134), Arg with Fmoc (134), Trp with formyl (134), and Ser, Thr, and Tyr with Ac (134). These groups expectedly withstand repeated acidolytic detritylations during the ODN synthesis and are removed by ammonolysis together with the nucleobase acyl protections. Met-containing conjugates are obtained by introducing methionine as a sulfoxide and reducing it back to methionine postsynthetically (141). Examples of the use of a hydroxylbearing linker between the ODN and peptide moieties include assembly of the peptide chain by the Boc chemistry on an ester linker and acylation of the N-terminus with 4-hydroxybenzoic

Reviews Scheme 16

acid (142) or 2,2-dimethyl-3-hydroxypropionic acid (143), peptide synthesis on an o-nitrophenylethanol linker followed byN-terminalacylationwithN-succinyl-6-aminohexanol(144,145), peptide synthesis on a 4-hydroxybutanoyl linker followed by N-terminal acylation with 4-[(6-hydroxyhexyl)amino]-4-oxobutanoic acid (146), and use of 10-hydroxydecanoyl group both as a support-bound linker and as an intervening spacer (147, 148). Alternatively, Fmoc chemistry may be applied to the initial assembly of the peptide chain. In a rather recent report exploiting this overall strategy, homoserine has been used as a connector between the peptide and ODN moieties (149). While the homoserine residue provides the hydroxyl function required as the starting point for the ODN chain assembly, it simultaneously offers an amino function to be used for fluorescent labeling. 6-Aminohexanol esterified to a succinyl linker has been used as a handle for the peptide synthesis, and the side chains have been protected with base-labile protecting groups: Tfa for Lys, Dmab for Asp, and 2-ClTr for Tyr (Scheme 10). NR-Fmocasparagines have been coupled as pentafluorophenyl esters without amide protection to avoid the side reactions that the HATU activation induces. Coupling of NR-Fmoc-O-Tr-homoserine to the N-terminus, fluorescent labeling of the deprotected amino group, and ODN chain assembly on the detritylated hydroxyl group, followed by conventional ammonolysis, complete the synthesis. Earlier, a related strategy involving normal


Fmoc peptide synthesis on a hydroxy-functionalized support, followed by coupling of 10-hydroxydecanoyl (150), 4-hydroxybutanoyl (151, 152), or 4-(6-hydroxyhexylamino)-4-oxobutanoyl (152) group as an intervening linker between the peptide and ODN moiety, has been applied. Very recently, a 35-mer peptide containing 25 trifunctional residues has been synthesized on a Rink-MBHA amide resin by the normal Fmoc/tBu chemistry and elongated with 3-trityloxypropionic acid. After acidolytic detritylation, an oligothymidylate sequence has been assembled by phosphoramidite chemistry. Consecutive treatments with piperidine in DMF and 95% TFA have then given the conjugate (153). The question that remains to be answered is whether a heterosequence withstands without depurination the acidolytic removal of the tBu-derived side-chain protecting groups. Although the stepwise synthesis of oligonucleotide-peptide conjugates is usually initiated by assembly of the peptide moiety, the opposite is also possible. The use of conventional Fmoc chemistry for the synthesis of the peptide moiety is, however, hampered by the fact that repeated removals of the Fmoc groups with organic bases also remove the 2-cyanoethyl protections from the internucleosidic phosphodiester linkages. To avoid this, an acid-labile 2-(biphen-4-yl)propan-2-yloxycarbonyl group (Bpoc) has been introduced for the R-amino protection (Scheme 11) (154). This group may be removed under the same conditions as used for removal of the 5′-O-DMTr groups during the ODN synthesis. MMTr protected 5′-amino-5′-deoxythymidine has been coupled as the last nucleoside to provide the amino group required for the peptide synthesis. Acyl protections can be used for both the amino acid side-chains and the nucleic acid bases, and hence, release from support and global deprotection is achieved by conventional ammonolysis. In spite of instability of the 2-cyanoethyl phosphate protections to Fmoc removal, Fmoc chemistry has been applied to the synthesis of 5′-peptide conjugates of ODNs on a single support (155). Up to octapeptide conjugates have been obtained by this approach. The acid-labile side-chain protections, viz., PhiPr for Asp and Glu, MMTr for Lys and Cys, Tr for Gln and His, and ClTrt for Tyr, are removed with TCA in DCM before ammonolysis. Compatibility with incorporation of arginine has not been tested. Previously, an essentially similar protocol has been applied to synthesis of up to tetrapeptide 5′-conjugates (156). Another possible way to avoid the problems arising from incompatibility of the 2-cyanoethyl phosphate and NR-Fmoc protections is the use Pd0-labile allyl group instead of 2-cyanoethyl group for protection of the phosphodiester linkages. Peptide-ODN hybrids consisting of two terminal ODN sequences and an intervening peptide segment have been prepared by this strategy (157). The 3′-ODN segment is first assembled on a sarcosine linker by the phosphoramidite chemistry, using 5′-N-MMTr-5′-amino-5′-deoxythymidine 3′-allyl-N,N-diisopropylphosphoramidite as the last building block (Scheme 12). The peptide sequence is then assembled on the deprotected amino group by the Fmoc chemistry, and the 5′-terminal ODN sequence is assembled on the terminal amino group. The allyl protecting groups and sarcosine linker are compatible with the Fmoc peptide chemistry when DBU is used for the removal of the Fmoc group. It is, however, worth noting that the feasibility of the approach has not been demonstrated with trifunctional amino acids. 3′,5′-Dipeptidyl conjugates of ODNs bearing very short peptide segments have been synthesized similarly (158, 159). Peptide nucleic acids (PNA), isosteric analogues of oligonucleotides where the nucleic acid bases are linked through N-(1oxoethylene) bridges to a peptide-like backbone composed of N-(2-aminoethyl)glycine units (160), are biologically stable and hybridize more efficiently and selectively than oligonucleotides, and they, hence, show potential as therapeutic oligonucleotide analogues. Unfortunately, they exhibit low cellular uptake and


Lo¨nnberg

Scheme 17

Chart 9

are unable to activate RNaseH, an enzyme that cleaves the RNA component of an mRNA/antisense oligomer duplex, providing the antisense oligomer with turnover (161). In addition, PNAs often suffer from solubility problems (161). Accordingly, conjugation of PNA with ODNs is not of any help for the targeting and internalization of ODNs. With PNA-ODN conjugates, the ODN moiety may rather be regarded as a conjugate group, which increases the solubility of PNA and

provides it with the ability to activate RNaseH. 5′-Terminal PNA-ODN conjugates have been prepared as described above for 5′-peptide conjugates, viz., by introduction of 5′-amino-5′deoxythymidine at the 5′-terminus of the support-anchored ODN and subsequent elongation of the chain by the PNA chain assembly (162). The 3′-conjugates have, in turn, been synthesized by using PNA building blocks bearing benzhydryloxycarbonyl (Bhoc) protection at the amino functions of the nucleic

Reviews


Scheme 18

acid bases (163). The key point is that, after the assembly of the PNA sequence by the Fmoc chemistry, the Bhoc groups are removed with TFA and replaced with conventional benzoyl protections on the support (Scheme 13). Finally, the terminal Fmoc group is removed, and the ODN chain is assembled on the exposed amino group. Cyclic ODN-PNA chimeras have been synthesized on an O-(2-chlorophenyl)-N-(6-aminohexyl)phosphoramidate linker (164). PNA is first assembled by Fmoc chemistry on the amino group of the linker and elongated with 6-DMTrO-hexanoic acid (Scheme 14). Owing to repeated removal of the Fmoc group with piperidine, the phosphodiester linkage becomes deprotected. The ODN moiety is then synthesized on the detritylated hydroxyl function, and the PNA-ODN hybrid is cyclized on support by treatment with MSNT in pyridine. Normal ammonolysis releases the cyclic conjugate and removes the base moiety protections. Fragmental coupling offers an alternative for the stepwise synthesis on a single support: a prefabicated peptide bearing a single unprotected amino group and base-labile side-chain protecting groups is conjugated to a support-bound oligonucleotide. Usually, the oligonucleotide is first assembled on the support and an appropriately activated peptide is linked to the 5′-terminal hydroxyl function. One possible activation method is conversion of the peptide to a phosphoramidite ragent. Normal

tetrazole activated coupling and subsequent oxidation to phosphoramidate is then carried out. The C-terminal amido nitrogen (165) and the side-chain hydroxyl group of a serine residue (166) have been used as a site of the phosphoramidite activation with 2-cyanoethyl-N,N-diisopropylphosphorochloridite. Both 3′- and 5′-conjugates have been prepared (166). 3′-Conjugates have been obtained by assembling the ODN chain in the inverse 5′f3′ direction and coupling the peptide phosphoramidite to the 3′terminal hydroxy function of the support-bound chain (Scheme 15). On using conjugation through the serine side chain, deprotection under drastically basic conditions may result in elimination of the ODN as a 5′-phosphate or 3′-phosphate by concomitant conversion of the serinyl residue to dehydroalanine. To avoid this, the phosphate linkages and the C-terminal carboxy function of the peptide have been protected with allyl groups and the nucleoside amino groups and the N-terminal amino group with Alloc groups. In an alternative approach, an R,ω-alkanediamine is tethered to the 5′-hydroxy function by 1,1′-carbonyldiimidazole activation and a preprepared protected peptide is coupled to the amino group by PyBOP/HOBt-promoted peptide bond formation (167). Up to dodecyl peptides have been successfully coupled. A long C12 spacer between the peptide and ODN and, interestingly, removal of the 2-cyanoethyl protections have improved the


Lo¨nnberg

Scheme 19

coupling yield (168). More recently, 1,6-diisocyanatohexane has been utilized to tether peptides bearing an N-terminal β-alanine to the 5-amino-3-oxapentyl tail of a protected support-bound ODN (169). The ε-amino functions of the peptides have to be protected with trifluoroacetyl groups, but no other side chain protections are necessary. On using the peptide in 10-fold excess, coupling yields up to 20% are achieved. Another feasible conjugation method depends on conversion of the 5′-aminoalkyl tail to an active urethane by reaction with bis(N-succinimidyl)carbonate (170). Up to 18-mer peptides have been attached in this manner to ODN phosphorothioates (Scheme 16). Instead of a 5′-aminoalkyl tail, the N-(3-aminopropanoyl) sidearm of a 2′-amino-2′-deoxy-arabino nucleotide within an otherwise protected support-bound oligonucleotide has been used as the site of conjugation (171). Unprotected peptides up to an heptamer have been conjugated by TBTU/HOBt coupling to solid-supported ODNs incorporating either a 2′-O-carboxymethyluridine unit (172) or a 5′carboxyalkyl tail (173). 80% yields have been obtained using the peptide in 100-fold excess. The carboxymethylated unit can be introduced as a phosphoramidite having the carboxy group protected as an Pd0-labile allyl ester and the 5′-carboxy group as a 1-methyl-4-oxo-4-trityloxybutyl phosphoramidite. An NFmoc-O-Tr-homoserinyl linker attached via an amide linkage to an amino-functionalized resin has been exploited in prepara-

tion of 3′-oligolysine conjugates by fragment coupling (40). The Fmoc group is first removed, and a trifluoroacetyl protected oligolysine is coupled. After that, the trityl protection is removed, and the ODN chain is assembled. Reaction of an aldehyde with hydrazine or alkoxyamine is one of the conjugation methods most extensively applied in the solution phase (120). The same method has also been successfully applied to solid-supported conjugation. Laminin peptide (H-RSGIYGPD-OH) has been conjugated as a C-aminooxy or hydrazine derivative to an aldehyde-functionalized ODN on a solid support (174). For this purpose, an appropriately protected 2′-O-[2,3-bis(allyloxycarbonyloxy)propyl]cytidine phosphoramidite building block has been introduced into the ODN sequence, the Pd-labile Alloc groups have been removed onsupport, and the resulting 2′-O-(2,3-dihydroxypropyl) group has been oxidized with periodate ion to a 2′-O-(2-oxoethyl) group. Treatment with a 1000-fold excess of the peptide conjugate and subsequent ammonolysis have given the conjugates in high yield.

GLYCOCONJUGATES Carbohydrate-protein interactions play a central role in cellular recognition. Usually, multiantennary carbohydrates anchored to proteins or lipids of cell membranes recognize

Reviews


Chart 10

extracellular proteins, but the opposite is also possible (175-177). For example, hyaluronic acid, a linear polysaccharide composed of a repeating disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosamine, [f3)-β-D-GlcNAc-(1f4)-β-D-GlcUA(1f]n, is the main ligand for a transmembrane glycoprotein CD44 that is overexpressed in many cancers (178), and hyaluronic acid conjugates of cancer drugs have been shown to exhibit increased uptake to cancer cells (179). Galactosylated PEG-conjugates of ODN phosphorothioates have, in turn, been reported to show enhanced uptake via the galactose-specific asialoglycoprotein receptor-mediated endycytosis (30), and antigenic peptides have been targeted to dendritic cells by an appropriate glycoconjugation (180). Additionally, it has been shown that galactosylated dendrimers are able to inhibit at nanomolar concentrations cellular binding of pathogens bearing galactose-binding membrane proteins (181). Accordingly, several lines of evidence suggest that glycotargeting may well be a viable method with which to enhance the delivery of therapeutic oligonucleotides (182). Preparation of oligonucleotide glycoconjugates has previously been reviewed in 2004 (183). In the present survey, attention is paid only to solidphase methods and mainly to those published since 2004. The protocols reported so far for the solid-phase synthesis of oligonucleotide glucoconjugates utilize phosphoramidite coupling, Cu(I) promoted 1,3-dipolar cycloaddition (“click reaction”), or oximation. Among these approaches, the phosphoramidite coupling is oldest, but still extensively applied. The simplest example is offered by introduction of a single monoor disaccharide unit into a terminal position of a support-bound ODN. To obtain 5′-glycosylated conjugates, appropriately

protected methyl glycosides have been converted to phosphoramidite reagents and introduced into the ODN by the last coupling cycle (Scheme 17) (184, 185). Besides sugar hydroxyl functions, the ω-hydroxy group of appropriately protected ω-hydroxyalkyl glycosides has been used as the site of phosphitylation (186, 187). 3′-Terminal sugar conjugates have, in turn, been prepared by assembling the ODN chain on the primary hydroxyl group of an O-acetyled glycoside immobilized to the support via a succinyl linker (184, 185). Conjugates bearing a sucrose moiety at both termini have been obtained by using these two approaches in combination (188). The 3′terminal sugar tail has been elongated by coupling one O6DMTr protected sugar phosphoramidite building block to the support-anchored sugar before the assembly of the ODN chain (189). A phosphoramidite building-block derived from methyl 4′-deoxy-β-D-lactoside (44; Chart 9) has been used to introduce a sugar moiety within the ODN chain (190). A spruce-like tetramannosyl conjugate has been prepared by using phosphoramidate 45 for branching, 46 for elongation of the branches, and 47 for the terminal mannosylation (191). In addition to mono- and disaccharides, more complicated carbohydrate structures have been conjugated to oligonucleotides by the phosphoramidite chemistry. Very recently, solid-phase synthesis of aminoglycoside conjugates bearing two neamine or ribostamycin units at an intrachain position has been described (192). The procedure is outlined in Scheme 18. The oligonucleotide chain is assembled by conventional phosphoramidite chemistry. At the desired site of aminoglycoside conjugation, an abasic ribofuranose phosphoramidite bearing one or two levulinoyloxyalkyl side arms is introduced. The levulinoyl


Lo¨nnberg

Scheme 20

protecting groups are removed with hydrazinium acetate on support and the appropriately protected aminoglycosides are coupled to the exposed hydroxyl functions as phosphoramidite reagents. An important detail of the synthesis is the protection of the aminoglycoside building block. Acyl groups readily migrate from hydroxyl functions to amino groups under basic conditions. That is why removal of the acyl protections from hydroxyl groups, while keeping the amino groups still protected, is highly desirable. Therefore, levulinoyl groups have been used instead of more common acetyl groups for protection of the hydroxyl functions. These groups may be removed under conditions that leave trifluoroacetyl protections on the amino groups intact. The latter protections are then removed upon ammonolysis used for the global deprotection and release of the conjugate from the support. Other complex structures conjugated to oligonucleotides by the solid-supported phosphoramidite chemistry include a trigalactosylated tetrahydroxycholane, introduced into the 3′-terminus of oligonucleotides with the aid of a branched linker (48) and to the 5′-terminus as a phosphoramidite reagent (49) (193), and 3′-(β-cyclodextrin) conjugates obtained by assembling the ODN

chain in the reversed 5′ f 3 ′ and conjugating an aminofunctionalized cyclodextrin to the terminal 3′-hydroxy function activated as a 4-nitrophenyl carbonate (44). Cyclodextrin conjugates have also been obtained by applying the phosphotriester conjugation method outlined in Scheme 3 in the foregoing (62). 3′-Conjugates derived from a tetrasaccharide glycomimetic have been prepared by sequential peptide and phosphoramidite coupling on a single support (194). An example is outlined in Scheme 19. An acetylated 1-azido-1-deoxy-6′O-DMTr-β-D-lactose is immobilized via a 4′-O-succinyl linker to an amino-functionalized support. The DMTr protection is converted to an acetyl protection and the azido group reduced with 1,3-propanedithiol. The resulting amino group is then subjected to peptide coupling with fully acetylated 1-azido-1deoxy-β-D-lacturonic acid using HATU/HOBt/DIPEA activation. The azido function is again reduced to an amino group, and a DMTr protected 6-hydroxyheptanoic linker is coupled. The ODN chain is then assembled on the detritylated hydroxyl function of this linker. Conventional ammonolysis gives the fully deprotected glycoconjugate.

Reviews Scheme 21

Chart 11

The most frequently used method for incorporation of sugars into intrachain positions of oligonucleotides is the use of phosphoramidites derived from prefabricated nucleoside conjugates. The building blocks successfully incorporated include 5′-O-DMTr protected 3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite)s of 5-[6-(2,3,4,6-tetra-O-acetyl-β-D-galactopyra-


nosyloxy)-1-hexynyl]-2′-deoxyuridine (50; Chart 10) (195, 196), 5-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)methyl-2′-deoxyuridine (51) (197), 5-[2,6-bis(trifluoroacetamido)-3,4-di-Oacetyl-2,6-dideoxy-β-D-glucopyranosyloxy]methyl-2′-deoxyuridine (52) (198), and 5-(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyloxy)methyl-2′-deoxyuridine (53) (199). This approach has been applied to introduction of even very complex carbohydrate groups into ODNs. For example, an aminoglycoside antibiotic, neomycin, has been incorporated into oligonucleotides as a C5-tethered 2′-deoxyuridine 3′-phosphoramidite building block (54) (200). An exceptionally long reaction time (30 min) was, however, required to couple this modified block. The Boc protections were removed after ammonolytic release in solution phase with a mixture of 3% TFA and 1% m-cresol in dioxane. It should be noted that the ODN chain contained no purine nucleosides prone to acid-catalyzed cleavage of the N-glycosidic bond. Several contiguous N-acetyl-D-galactosamine groups have been introduced into ORNs with the aid of a phosphoramidite reagent derived from N-[12-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-βD-galactopyranosyl)dodecanoyl]threoninol (201). Click chemistry has recently gained popularity in preparation of bioconjugates. Several applications concerning solid-supported synthesis of oligonucleotide glycoconjugates have also been reported. Usually, sugar azides are conjugated to support-anchored alkynylated oligonucleotides. ODNs bearing up to three β-Dgalactopyranosyl or R-D-mannopyranosyl groups at the 3′-terminus have been obtained by consecutive application of oxidative amidation and microwave-assisted click chemistry (202, 203). Scheme 20 outlines the procedure. A non-nucleosidic tail consisting of cyclohexane-1,4-diyldimethanol or tetraethylene glycol units is first assembled by H-phosphonate chemistry, and the H-phosphonate diester linkages obtained are subjected to amination with propargylamine. Fully acetylated 8-azido-3,6-dioxaoctyl glycopyranosides are then attached by Cu(I)-promoted 1,3-dipolar cycloaddition under microwave irradiation. The ODN sequence is assembled by the phosphoramidate chemistry on the hydroxyl function of the last non-nucleosidic unit, and conventional ammonolysis completes the synthesis. Another indication of the applicability of click chemistry to the solid-supported construction of oligonucleotide glycoconjugates is the synthesis of fucosylated pentaerythrityl phosphodiester oligomers (204). Very recently, an example of a reaction of an alkynylated sugar with a support-bound azido-functionalized oligonucleotide has been reported (205). A 5′-DMTr protected 3′H-phosphonate monomer derived from 4′-C-azidomethylthymidine has been prepared and inserted into an ODN by the H-phosphonate chemistry (Scheme 21). The corresponding phosphoramidite building block cannot be prepared, owing to an intramolecular Staudinger reaction between the azido and phosphoramidite moieties. The unmodified nucleosides may, however, be coupled as phosphoramidites even though 4′-C-azidomethylthymidine blocks would have already been incorporated into the chain. While the click reaction has been observed to be virtually quantitative, removal of the copper complex of tris[(1-benzyl-1,2,3-triazol-4-yl)methyl]amine (TBTA) used as a promoter requires special attention, as indicated by capillary electrophoresis of the released product before and after HPLC purification. Solid-phase oximation has been applied to preparation of ODN 5′-glycoconjugates bearing up to 6 identical monosaccharides (206) For this purpose, a non-nucleosidic phosphoramidite building block containing two phthaloyl protected aminooxy functions has been prepared and inserted into an ODN by the phosphoramidite chemistry. These protections may be easily removed on a solid support, and sugars may be introduced by oximation with alkyl glycosides bearing an aldehyde function on the aglycon (Scheme 22).


Lo¨nnberg

Scheme 22

The oxime linkages withstand standard ammonolysis, which also quantitatively removes the acetyl protections from the sugar hydroxyl functions. Usually, lectins bind only one type of sugar, and hence, homoclusters are considered to adequately guarantee high affinity binding (175, 177). There are, however, some indications that in special cases heteroclusters containing more than one type of sugars bind more tightly than the corresponding homoclusters (207). For example, mixed-type R-Man, β-Glc and R-Man, β-Lac heteroglycoclusters exhibit 8 times higher affinity to Con A than R-Man homoclusters of identical valency (208). Accordingly, synthesis of oligonucleotide glycoconjugates containing more than one type of sugar ligands is of interest. To construct such conjugates on a solid support, a non-nucleosidic bis(hydroxymethyl)malondiamide-based building block bearing three orthogonally removable hydroxy protections has been prepared and introduced into the 5′-terminus of a solid-supported ODN (209). The hydroxyl protections are removed in a stepwise manner, and the sugar units are introduced as phosphoramidite reagents. Methyl-protected phosphoramidites have to be used for the chain assembly, since the 2-cyanoethyl groups

do not withstand removal of the fluoride ion labile TBDPS group. For the same reason, a polymer support has to be used instead of CPG. As depicted in Scheme 23, the first sugar unit is attached after detritylation. Then, the levulinoyl protection is removed and the second sugar phosphoramidite is coupled, and removal of TBDPS with triethylamine hydrogen fluoride followed by coupling of the last sugar completes the conjugation process. The phosphodiester likages are demethylated before normal ammonolysis. Solid-supported glycosidation has very seldom used for the preparation of oligonucleotide glycoconjugates on a single solid support. Glycosidation usually depends on electrophilic activation, and this easily results in depurination of the ODN chain. Very short 5′-glycosylated conjugates of ODNs have, however, been obtained by glycosylating the 5′-terminal hydroxyl function of the support-bound ODN with fully benzoylated glycopyranosyl trichloroacetimidate using trimethylsilyl triflate as an activator (210, 211).

MISCELLANEOUS Conjugation of ODN phosphorothioates with poly(ethylene glycol) polymers ranging from 6 to 20 kDa has been shown

Reviews Scheme 23

to increase their half-life in rat plasma, to enhance the stability against nucleases, and to improve the pharmacokinetics (31). 3′-Terminal ODN conjugates of poly(ethylene glycol)s with degrees of polymerization up to 150 have been prepared by immobilization of the PEG oligomer to an aminoalkylated support via a succinyl linker and subsequent synthesis of the ODN chain by the phosphoramidite (212-214) or H-phosphonate chemistry (215). N-Fmoc protected aminopoly(ethylene glycol)-900 (216) and PEG polymers up to the length of 120 units (213, 214) have been conjugated as phosphoramidite reagents to the 5′-terminus of a supportbound oligonucleotide. Among small molecules known to undergo receptor-mediated cellular uptake, folic acid has received interest as a potential carrier of oligonucleotides into cells. 3′-Terminal folic acid conjugates have been prepared with the aid of a branched linker, as depicted in Scheme 24 (217), and intrachain conjugates by insertion of an appropriately protected non-nucleosidic phosphoramidite reagent

Bioconjugate Chem., Vol. 20, No. 6, 2009 1087 Scheme 24

(55; Chart 11) into the desired place of the chain (201). Puromycin, porphyrins, groove binders, and intercalators have also received considerable interest as conjugate groups. Although these conjugates are not primarily aimed at exhibiting enhanced cellular uptake or cell-type specific targeting, their solid-phase synthesis is briefly discussed here. 3′-Conjugates of puromycin have been obtained by conventional ODN synthesis on puromycin-derived linker 56 (218, 219). Porphyrins have been conjugated as nucleoside derived phosphoramidite (220) or hydrogen phosphonate (221) building blocks, as non-nucleosidic phosphoramidite (222) or hydrogen phosphonate (223, 224) reagents, or as carboxy-functionalized derivatives to the 5′-amino group of a support-bound ODN (225). Groove binders have been coupled as pentafluorophenyl esters to a 5′-amino tail of a support-bound ODN (226, 227) or to an amino group of a support-bound linker before the ODN chain assembly (227, 228), or they have been coupled as an alcohol to the 5′-terminal OH activated as 2-cyanoethyl N,N-diisopropylphosphoramidite (229). Intercalators are introduced as non-nucleosidic phosphoramidite reagents (230, 231) or prefabricated nucleosidederived building blocks (230), or they are attached as an active ester to an amino group of a branched linker (233, 234).

LITERATURE CITED (1) Sasaki, S., and Nagatsuki, F. (2006) Application of unnatural oligonucleotides to chemical modification of gene expression. Curr. Opin. Chem. Biol. 10, 615–621.

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