MAY/JUNE 2005 Volume 16, Number 3 © Copyright 2005 by the American Chemical Society
REVIEWS Use of Carbonyl Group Addition-Elimination Reactions for Synthesis of Nucleic Acid Conjugates Timofei S. Zatsepin,† Dmitry A. Stetsenko,‡,§ Michael J. Gait,‡ and Tatiana S. Oretskaya†,* Department of Chemistry, M. V. Lomonossov Moscow State University, 1 Leninskie Gory, Moscow, 119992, Russia, and Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Received December 2, 2004; Revised Manuscript Received March 3, 2005
This review outlines the synthesis of covalent conjugates of oligonucleotides and their analogues that are obtained by reactions of carbonyl compounds with various nucleophiles such as primary amines, N-alkoxyamines, hydrazines, and hydrazides. The products linked by imino, oxime, hydrazone, or thiazolidine groups are shown to be useful intermediates for a wide range of chemical biology applications. Methods for their preparation, isolation, purification, and analysis are highlighted, and the comparative stabilities of the respective linkages are evaluated. The relative merits of incorporation of a carbonyl group, particularly an aldehyde group, into either the oligonucleotide or the ligand parts are considered. Examples of harnessing of aldehyde-nucleophile coupling for the labeling of nucleic acids are given, as well as their conjugation to various biomolecules (e.g. peptides and small molecule ligands), site-specific cross-linking of oligonucleotides to nucleic acid-binding proteins, assembly of multibranched supramolecular structures, and immobilization on functionalized surfaces. Future perspectives of bioconjugation and complex molecular engineering via carbonyl group additionelimination reactions in nucleic acids chemistry are discussed.
INTRODUCTION
Structurally diverse conjugates of oligonucleotides have found numerous applications in different fields of molecular and cell biology. Molecules attached to oligonu* To whom correspondence should be addressed. Phone: +7(095) 939-5411. Fax: +7(095) 939-3181. E-mail: oretskaya@ belozersky.msu.ru. † MSU, Russia. ‡ MRC, UK. § Present address: School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
cleotides are often used as reporter groups, e.g. fluorescent (1, 2), electrochemical (3, 4), or spin (5-7) labels, for stabilization of double- or triple-helical structures, e.g. intercalators (8) and minor groove binders (9, 10), and for increase of cellular uptake of conjugates, e.g. peptides (11-13), hydrophobic molecules (14), and certain carbohydrates (15). There are two alternative approaches to the synthesis of modified nucleic acids: (a) automated solid-supported phosphoramidite or H-phosphonate oligonucleotide assembly using appropriately modified nucleoside or nonnucleoside derivatives (16-18), and (b) enzymatic incorporation of a 5′-triphosphate of a modified nucleoside (19).
10.1021/bc049712v CCC: $30.25 © 2005 American Chemical Society Published on Web 05/04/2005
472 Bioconjugate Chem., Vol. 16, No. 3, 2005 Scheme 1. Reductive Amination: Schiff Base (imine) Formation Followed by Reduction
Chemically synthesized oligonucleotides may be extended by polymerase chain reaction (PCR) or joined by chemical (20) or enzymatic (21) ligation. The triphosphate approach provides an opportunity for multiple incorporation of modified nucleoside 5′-triphosphate derivatives into either DNA or RNA. Unfortunately, this method is limited mainly to base-modified nucleoside derivatives, since most nucleoside modifications lead to an inability of the corresponding 5′-triphosphate to be incorporated by polymerases (22). Three strategies are generally used for synthesis of oligonucleotide conjugates. The first approach requires synthesis of a nucleoside or non-nucleoside synthon followed by appropriate protection and phosphitylation. The phosphoramidite or H-phosphonate derivative is then incorporated into an oligonucleotide chain during solid-phase synthesis. The conjugated molecule needs to be stable during oligonucleotide synthesis and deprotection. A limitation of this approach is that a reasonably large-scale and multistep synthesis of a modified monomer is required. It seems to be optimal only for incorporation of those reporter or marker groups that are chemically robust and easily accessible synthetically. The second approach employs fragment conjugation of a target molecule to fully protected support-immobilized oligonucleotide containing a free reactive group. This reactive function must be blocked with a protecting group that can be removed without affecting other protecting and anchoring groups of the oligonucleotide. Selective deprotection of the reactive group is followed by conjugation and then by cleavage and deprotection of the conjugate. Only certain selectively removable protecting groups/conditions for their removal have been used for preparation of oligonucleotide conjugates, such as allyloxycarbonyl (Alloc1)/Pd (0) (23, 24), fluorenylmethyloxycarbonyl (Fmoc)/morpholine (25, 26), levulinoyl (Lev), and phthaloyl (Phth)/hydrazinium acetate (24, 27), and photolabile protecting groups of the 2-nitrobenzyl type (28, 29). In some cases, preliminary deblocking of internucleoside phosphates is needed to increase the solvation of the oligonucleotide chain and to remove reactive acrylonitrile that sometimes causes side-reactions. The approach is indispensable for synthesis of combinatorial libraries, especially in the case of hydrophobic ligands, since a large excess of the ligand can be used (30). However, the resultant conjugate also needs to be stable under the deprotection conditions. 1 Alloc, allyloxycarbonyl; AP site, abasic, or apurinic/apyrimidinic, site; Bn, benzyl; Boc, tert-butoxycarbonyl; Bz, benzoyl; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DEAD, diethyl 1,1′azodicarboxylate; DEC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; DMTr, 4,4′-dimethoxytrityl; Fmoc, 9-fluorenylmethoxycarbonyl; Lev, levulinoyl (4-oxopentanoyl); MMTr, 4-methoxytrityl; OSu, N-hydroxysuccinimide ester; Phth, phthaloyl; Py, pyridine; RP-HPLC -reverse-phased HPLC; SNP, single nucleotide polymorphism; TBDMS, tert-butyldimethylsilyl; Tf, trifluoromethanesulfonyl; Tr, trityl (triphenylmethyl).
Zatsepin et al. Scheme 2. Reductive Amination of an Apurinic Site with an Amine under Neutral or Acidic pH
In the third approach, conjugation of a deprotected and (if necessary) purified oligonucleotide is carried out in solution. This approach seems to be the most flexible, since no harsh treatment is required after the reaction. The main problem is the need for isolation of the conjugate from the reaction mixture. There are many different types of chemistry available for fragment coupling in solution. Oligonucleotides with amino or thiol groups are conjugated by amide, disulfide, or thioether bond formation (16), while those with electrophilic groups are rarely used because of the complicated synthesis of the appropriate derivatives (31). However in the case of complex biomolecules, regio- and chemoselectivity are the main concerns in the reaction. Chemoselective conjugation is widely used in peptide chemistry (32, 33), carbohydrate chemistry (34), chemistry of nucleic acids (35), and steroids (36-38). During the past decade, a number of classical organic reactions, such as the Staudinger (39, 40), Diels-Alder (41-43), and 1,3-dipolar cycloaddition reactions (44-46), have been applied successfully to the bioconjugation of nucleic acids. Extraordinary regioselectivity and high yields of the reactions make this approach the method of choice for biomolecular conjugation. Here we describe the application of addition-elimination reactions of a carbonyl group, mainly an aldehyde group, with various nitrogen nucleophiles for chemoselective conjugation of the appropriately modified oligonucleotides. We discuss both the introduction of an aldehyde group into an oligonucleotide as well as into the molecule to be conjugated. CONJUGATION VIA IMINE, OR SCHIFF BASE FORMATION FOLLOWED BY BOROHYDRIDE REDUCTION
Schiff base, or imine, formation followed by reduction, i.e. reductive amination, is widely used in total synthesis of natural products. The reaction is slower at low pH due to protonation of the amino group but may be carried out at neutral or mildly basic conditions. However, the reaction is reversible, so the imine should be reduced in situ to the secondary amine to drive it to completion (Scheme 1). Various borohydride reagents are used for the reduction, but sodium borohydride and sodium cyanoborohydride are by far the most common. Sodium cyanoborohydride is more stable in aqueous solution and is a milder reductant and thus is used more frequently. Methods of synthesis of both amino (16, 18) and aldehyde (31) oligonucleotides are reviewed elsewhere. Here we would like to outline the conditions of the
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Reviews
Scheme 3. 1,4-Addition of 3-Aminocarbazole to 2,3-Dideoxy-2,3-dehydroribose Residue at the 3′-End
Scheme 4. Reductive Amination of 3′-Terminal Periodate-Cleaved RNA
Scheme 5. Aldehyde Derivatives of Oligonucleotides That Have Been Used for Reductive Amination
Scheme 6. Aldehyde Derivatives of Oligonucleotides Used for Affinity Modification of DNA-Binding Proteins via Reductive Amination
Scheme 7. Modified Nucleoside and Dinucleosides Used for Template-Directed Polymerization
reaction and emphasize the influence of both the nature and placement of an aldehyde group on the conjugate yield. Probably, the most common example of reductive amination via Schiff base formation is aminooligonucleotide immobilization on various aldehyde-coated surfaces for DNA chip fabrication (47). This combination of reactive groups is more effective then the opposite due to nonspecific interactions between anionic oligonucleotides and positively charged surface (48). However in the case of aldehyde-coated surfaces, blocking of the unreacted aldehyde groups by aminooligoethyleneglycol decreases nonspecific oligonucleotide binding and provides
better sensitivity during hybridization assays (49-50). Another reason for immobilization of aminooligonucleotides on the aldehyde surfaces has been the lack until recently of commercial availability of aldehyde phosphoramidites. Nevertheless, two common methods for the
474 Bioconjugate Chem., Vol. 16, No. 3, 2005 Scheme 8. C-Terminal PNA Aldehyde
introduction of an aldehyde group into nucleic acids have been used. The most popular method for generation of an aldehyde group in native DNA is partial acidic depurination. Proudnikov et al. (51) demonstrated that DNA that contains apurinic, or AP, sites undergoes facile β-elimination under neutral conditions. However, reductive amination proceeds smoothly under slightly acidic pH (Scheme 2). Vasseur et al. (52) demonstrated that reaction of the AP site with 3-aminocarbazole leads to a chain break followed by nucleophilic addition to R,β-unsaturated
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aldehyde (Scheme 3). Such complex processes have not been followed step by step by others, but Greenberg et al. (53) later observed an analogous process during interactions of a lysine residue of DNA glycosylase with ribonolactone at the 3′-end of an oligonucleotide. Later Manoharan et al. (54) conjugated a number of amines, including tripeptides and aminophenanthroline, to an AP site in oligodeoxyribonucleotides under reductive conditions, without giving the yields of the conjugates. Another common method for generation of an aldehyde group in nucleic acids is periodate oxidation of the 3′terminal ribonucleotide in native RNA, synthetic oligoribo- or oligodeoxyribonucleotides 3′-modified with a single ribonucleotide residue. Apart from conjugation of the resulting dialdehyde derivatives with amines, some β-elimination also takes place (55) (Scheme 4). These approaches may be useful for immobilization of nucleic acids on a surface or on beads, where a large excess of oligonucleotide is normally used, but they are far from optimal for the synthesis of various oligonucleotide conjugates. However, Schiff base formation has been used to synthesize conjugates of oligonucleotides that contain an aldehyde group (Scheme 5) with alkaline phosphatase (56), methionine (57), 1-aminomethylpyrene, aminooligoethyleneglycol (58), and an aminoalkyl derivative of biotin (59, 60). Sando et al. (61) applied a different approach, the conjugation of 5′-amino oligonucleotides with reducing carbohydrates. Phosphate and borate buffers that cover a wide basic pH range (6.8-9.5) were used. The yields of the conjugates varied from 50% to
Scheme 9. Oligonucleotide Ligation by Metal-Directed Salen Complex Formation
Scheme 10. Phosphoramidites Used for Synthesis of Supramolecular Structures
Reviews Scheme 11. Synthesis of N-Hydroxyphthalimide Derivatives
quantitative, but a large excess of the amine is usually used. Sando et al. (61) emphasized the superiority of the borate buffer due to an increase in the reaction rate. Kittaka et al. used modified oligonucleotides bearing aldehyde groups (Scheme 6) in the heterocyclic base for affinity modification of Rel/NF-kB (62-64), c-Myb (65), or RecA proteins (66, 67). Schiff base formation with lysine residues significantly increased binding to the protein. However, oligonucleotides containing 6-formyl2′-deoxy- and 6-formyl-2′-O-methyluridine were found to be ineffective due to significant duplex disturbance and low binding to proteins. The duplex structure was somewhere between A- and B-forms, as judged by CDspectroscopy (65, 68). More recently, oligonucleotides containing an aldehyde group at the 2′-position of a sugar residue were used for affinity modification of NF-kB protein (69, 70) and DNA-methyltransferase SsoII (Vorobjeva, Zatsepin, Kubareva, Oretskaya, unpublished results). Also oligonucleotides with 2′-dialdehyde group were used in affinity modification of methyltransferases (71) and T7 RNA polymerase (72). Recently Bugaut et al. (73) used oligonucleotides containing 2′-amino-2′-deoxyuridine for identification of duplex-stabilizing ligands bearing an aldehyde group by a dynamic combinatorial library approach. A number of aromatic aldehydes were added to DNA duplexes containing a 2′-amino-2′-deoxyuridine residue at the 3′- or 5′-terminus or in the middle of an oligonucleotide chain. The ratio of conjugates as a function of time was
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monitored by HPLC. Imines formed were reduced to the corresponding amines, which are analyzed more easily. It was demonstrated that the portion of product containing the most stable conjugate is increased, and that the rate of change of that portion is proportional to duplex stability. During the past decade, interest in DNA templatedirected organic synthesis has increased significantly (21, 74-77). Earlier the main efforts were centered on chemical ligation of short oligonucleotides or monomeric nucleotides on a DNA template. Today DNA templatedirected synthesis allows for regioselective chemical reactions, otherwise ineffective or even impossible in aqueous solution. Lynn et al. (78, 79) used Schiff base formation to ligate modified oligonucleotides. It was demonstrated that the imine reduction shifts the equilibrium toward products and allows the reaction to function in a catalytic mode due to significant destabilization of the duplex (79). Later Lynn et al. demonstrated oligomerization of a modified thymidine or thymidine dinucleoside that carry 5′-amino and 3′-aldehyde groups (Scheme 7) on an oligoadenylate template (80, 81). It should be noted that the process was effective only if the template was in solution or immobilized on a polystyrene support, and was very sensitive to mismatches. Liu et al. (82-86) used 5′- or 3′-aldehyde oligonucleotides for ligation on a DNA template with aminooligonucleotides or cross-linking to a modified matrix. The authors studied the dependence of the reaction yield on the structure of the template i.e. its length, presence of loops and reactive groups. Reductive amination showed a pronounced distance-dependence in the case of linear templates, but a lower distance-dependence was observed for a looped template. A number of subsequent ligations and cross-linkings with different templates allowed the synthesis of rather complex molecules conjugated to DNA (e.g. macrocyclic compounds (85, 86)). Later Rosenbaum and Liu (87) carried out oligomerization of a PNA tetramer containing a C-terminal aldehyde group (Scheme
Scheme 12. Phosphoramidite Derivatives Obtained via Mitsunobu Reaction That Were Used for Synthesis of Aminooxyoligonucleotides
476 Bioconjugate Chem., Vol. 16, No. 3, 2005 Scheme 13. Synthesis of a Protected Aminooxy Building Block by Nucleophilic Subsitution
8) on a long DNA template (40 nt). The process could be described as an artificial DNA replication. Czlapinski and Sheppard (88, 89) applied metal-salen complex formation for oligonucleotide ligation on a DNA template or ligation of self-complementary oligonucleotides (Scheme 9). Oligonucleotides containing 5′- or 3′salicylaldehyde moieties, respectively, were ligated on a DNA template by ethylenediamine in the presence of Mn2+ or Ni2+. The yields were up to 74% (compared with aliphatic hydrazine > hydrazides.
Scheme 25. Double Conjugation to 3′,5′-Bis-Modified Oligonucleotides
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Scheme 26. 5′-Triphosphates of Ketonucleosides Used for Incorporation into RNA and the Aminooxy Fluorescein Derivative for Subsequent Labeling via Oxime Formation
Scheme 27. Keto Oligonucleotides and Aminooxy Compounds Used for Conjugation (144)
Scheme 28. Conjugation of a Keto Bis-PNA with an Aminooxypeptide
Since hydrazino/hydrazido group may be incorporated into either an oligonucleotide or a ligand, this section is divided into two parts covering both possible cases. Synthesis of Hydrazido Oligonucleotides and Their Conjugation with Aldehydes. Most of the oligonucleotides containing hydrazide groups have been synthesized by hydrazinolysis of ester groups in nucleosides or oligonucleotides during a hydrazine-mediated deblocking reaction. In 1989 Ghosh et al. (147) synthesized a 5′-hydrazide derivative by coupling carbohydrazide to an activated 5′-phosphate group of an oligonucleotide
Scheme 29. Hydrolysis of a Hydrazone Linkage
(Scheme 30). These oligonucleotides have been used to conjugate 2,4-dinitrobenzaldehyde hapten and aldehydemodified alkaline phosphatase. In the latter case, several oligonucleotides were attached to the enzyme. Oshevski (148) described synthesis of a 3′-hydrazide by phosphoramidate formation. The oligonucleotide containing a 4-aminomethylbenzhydrazide residue was ligated to a 5′-aldehyde oligonucleotide on a DNA template (Scheme 31). Usually the yields did not exceed 20% under optimal conditions, and no ligation was observed in the case of mismatches in a DNA template close to the ligation point. The system was proposed for SNP detection. Raddatz et al. (149) synthesized a series of blocked hydrazide phosphoramidites by hydrazinolysis of esters followed by phosphitylation (Scheme 32). Also several phosphoramidites containing ester groups were prepared, and hydrazide formation was carried out during oligonucleotide deblocking step. Some of the derivatives carried two or four hydrazide moieties. The hydrazide oligonucleotides were conjugated to a peptide and used for DNA array preparation. It was shown that effectiveness of immobilization increased as the number of hydrazide groups per oligonucleotide increased. Later von Kiedrowski et al. (150) prepared a branched oligonucleotide with a defined structure by hydrazone formation with 1,3,5-benzenetricarboxaldehyde on a branched DNA template (Scheme 33). Sodium cyanoborohydride reduction slightly increased the yield. No reaction occurred without template. Zubin et al. (151) attempted a synthesis of oligonucleotides containing a hydrazide group at the 2′-position of a sugar residue via a blocked uridine 2′-hydrazido phosphoramidite (Scheme 34). However, the authors reported only a conversion of the hydrazide group into the amide during ammonia treatment, even under milder conditions. This may be ascribed to the more electrophilic nature of the hydrazide.
Reviews
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Scheme 30. Oligonucleotides Containing 5′-Carbohydrazide and Their Conjugation to Aldehydes
Scheme 31. Template-Directed Ligation of the 3′-Hydrazide and 5′-Aldehyde Oligonucleotides
Scheme 32. Phosphoramidite Building Blocks Used for Synthesis of Hydrazide Oligonucleotides. Generation of a Hydrazide Group in the Modified Oligonucleotides
Gao and Orgel (152) used hydrazone formation for the synthesis of a rigidly cross-linked base pair within an oligonucleotide sequence (Scheme 35). Introduction of a hydrazino group into the 4-position of a cytosine residue by transamination in the presence of bisulfite was followed by conjugation with the modified nucleoside containing a malondialdehyde group. Heterocyclic bases of the cross-linked pair are coplanar, and the geometry of the pair resembles that of native double-stranded DNA. These properties allow the oligomerization of activated nucleotides on a template cross-linked to the starting nucleoside. The product of the oligomerization
reaction could be detached by an excess of hydrazine. Hydrazone reduction leads to a significant decrease in the yield of the ligation product. Zhao et al. (153, 154) demonstrated the application of commercially available 5′- and 3′-aldehydo or hydrazino oligonucleotides for DNA ligation, conjugation to modified antibodies or for immobilization on a modified glass surface (Scheme 36). This method for oligonucleotide immobilization was further used by Zhong et al. (155). The commercial availability (Solulink, San Diego, CA) of N-hydroxysuccinimide esters of 6-hydrazinonicotinic acid and 4-formylbenzoic acid and the corresponding
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Scheme 33. Ligation of Hydrazide Oligonucleotides with 1,3,5-Benzenetricarboxaldehyde on a Branched Tris-Oligonucleotide Template
Scheme 34. An Attempt To Prepare the 2′-Hydrazide Oligonucleotides via a Presynthesized 2′-Hydrazide Synthone
phosphoramidites that can be used for incorporation of an aldehyde or hydrazine group into oligonucleotides and other biomolecules may add significantly to the popularity of hydrazone coupling in nucleic acid chemistry. Conjugation of Aldehyde Oligonucleotides with Hydrazines and Hydrazides. A number of publications
describe conjugation of aldehyde oligonucleotides to hydrazines and hydrazides. Kremsky et al. (156) biotinylated a 5′-aldehyde oligonucleotide by hydrazone formation with biotin hydrazide. Later Sonveaux et al. (59, 60) used the same scheme for oligonucleotides containing an aldehyde group in the middle of a sequence. In both cases the conversion was quantitative under reducing conditions. Melnyk et al. (157) used 5′-glyoxylyl oligonucleotides for conjugation with a hydrazino peptide (Scheme 37). A 5′-amino oligonucleotide was acylated by O,O′-diacetyl tartaric anhydride on solid phase. A glyoxylyl group was generated by periodate oxidation after oligonucleotide deprotection. Very recently a different scheme was used for the synthesis of 5′-glyoxylyl oligonucleotides by Defrancq et al. (158). An O,N-blocked serine residue was introduced into oligonucleotides by use of a phosphoramidite during standard DNA synthesis, followed by ammonolysis and periodate oxidation (Scheme 37). The presence of an electron-withdrawing amide group placed R to the aldehyde increases its reactivity and the stability of the resultant hydrazone. It was shown that ca. 50% conversion occurs in a few seconds and the reaction was complete in 15 min (157). However, the stability of the aldoxime linkage
Scheme 35. Conjugation of N-Aminocytosine Oligonucleotide with Malondialdehyde Nucleoside
Reviews
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Scheme 36. Conjugation Reactions of Hydrazino and Aldehydo Oligonucleotides Prepared from a Commercially Available Set of Reagents
Scheme 37. Synthesis of 5′-Glyoxylyl Oligonucleotides and Conjugation with N-Terminal Hydrazino and Aminooxy Peptides
is lower at basic pH compared to other oxime linkages (158). Incorporation of an aldehyde group onto the 5′-end of an oligonucleotide limits the number and position of conjugated moieties. An aldehyde group in the 2′-position of ribose provides an opportunity for multiple conjugation of hydrazides and hydrazines, as has been shown by Zatsepin et al. (138). However, the hydrazones formed were prone to hydrolysis in the neutral and basic pH range, and borohydride reduction was required (Scheme 38). More recently, oligonucleotide-containing aldehyde groups in the 2′-position of one or more arabinouridine residues have been prepared (159). It was demonstrated that a hydrazone formed by an aromatic hydrazine, 9-hydrazinoacridine, is more stable in comparison to hydrazones of aliphatic hydrazides and does not need reduction in order to be purified by HPLC. Tetramethylrhodamine (TAMRA) hydrazine (Scheme 39) was used for DNA or RNA labeling through an AP
site or through a 3′-periodate-oxidized ribose (160), respectively, as described previously for aminooxy derivatives. Labeling was carried out under acidic pH followed by sodium cyanoborohydride reduction. Later Manoharan et al. (54) also used an AP site to incorporate hydrazide derivatives of biotin, pyrene, and fluorescein. These authors generated an AP site enzymatically by treatment of DNA with uracil-DNA glycosylase. Aldehyde oligonucleotides have been used many times for immobilization on hydrazide-modified surfaces. Kremsky et al. (156) and Schluep and Cooney (161, 162) used aldehyde oligonucleotides for immobilization on hydrazide-modified latex or glass, respectively (Scheme 40). Hydrazone reduction led to an increase in the extent of immobilization. Later glyoxylyl (163) and benzaldehyde (164) oligonucleotides were used for immobilization on a semicarbazide-modified glass (Scheme 41). Semicarbazide glass was synthesized either by hydrazine addition to the
484 Bioconjugate Chem., Vol. 16, No. 3, 2005 Scheme 38. Synthesis of Oligonucleotide-Peptide Conjugates via Hydrazone Formation Followed by Reduction
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homogeneity and higher density of the array. The hydrazones were rather stable over a wide pH range, but borohydride reduction again increased the shelf life of the DNA chips obtained. CONJUGATION VIA REACTIONS OF ALDEHYDE OLIGONUCLEOTIDES WITH MISCELLANEOUS NUCLEOPHILES
Scheme 39. TAMRA Hydrazine
Scheme 40. Immobilization of the 5′-Aldehyde Oligonucleotides on Hydrazide Surfaces
As it was discussed above, reaction of a side-chain amino group in amino acids with aldehydes leads to an imine formation. However in the case of cysteine, the reaction is followed by intramolecular addition of the thiol group with concomitant thiazolidine formation (Scheme 42). This approach was used to synthesize a number of oligonucleotide-peptide conjugates (111, 138, 141). An aldehyde group was introduced onto the 5′-end of an oligonucleotide or into the 2′-position of a sugar moiety. Zatsepin et al. (165) introduced a 2,3-diaminopropoxy group into the 2′-position of a sugar moiety and demonstrated a model conjugation with benzaldehyde via imidazolidine formation (Scheme 43). However, the imidazolidine is less stable than a thiazolidine both in basic and acidic conditions, so the application of the former has less general utility than that of the latter. Liu et al. published a number of inspiring contributions (82-84, 166, 167) on use of DNA as a template for organic reactions. These authors explored Wittig reaction, nitroaldol condensation (Henry reaction), or oxazolidine formation (Scheme 44). The reactions were carried out in aqueous medium. The driving force seems to be a spatial proximity of the reactive groups on oligonucleotides. The groups were incorporated at adjacent positions on complementary oligonucleotides or in contiguous oligonucleotides bound to a DNA template. It is evident that these methods would allow for adaptation of many types of reaction from classical organic chemistry for modification of nucleic acids. CONCLUSIONS
isocyanate glass (163) or via a direct silylation by the silane containing a blocked (163) or even a free (164) semicarbazide group. The first approach gave better
Analysis of the literature demonstrates that the most widely used approach that involves carbonyl group addition-elimination reactions for synthesis of oligonucleotide conjugates is still Schiff base (imine) formation followed by borohydride reduction. Usually the yields are not quantitative and a number of side reactions occur, but the accessibility of starting materials makes this approach rather popular. The method is especially effective for conjugation or immobilization of native DNA and RNA. However, in the case of chemically synthesized oligonucleotides the oxime and hydrazone formation are
Scheme 41. Synthesis of the Semicarbazide Glass and Immobilization of the 5′-Benzaldehyde or Glyoxylyl Oligonucleotides on It
Reviews Scheme 42. Conjugation of Aldehyde Oligonucleotides with Cysteine Derivatives via Thiazolidine Formation
Scheme 43. Conjugation of the 2′-(2,3-Diaminopropoxy) Oligonucleotides with Benzaldehyde
Bioconjugate Chem., Vol. 16, No. 3, 2005 485 Scheme 44. DNA Ligation via Wittig or Henry Reaction or Oxazolidine Formation
choices either in reagents or in chemical methods available to prepare nucleic acids conjugates that are purposefully constructed to meet the requirements of any particular biological application. ACKNOWLEDGMENT
The authors thank Drs. A. Yu. Tomashevsky, University of Virginia, Charlottesville, VA, and A. Zamyatnin, Swedish University of Agricultural Sciences, Uppsala, Sweden, for their valuable assistance in preparation of this review for publication. The review has been written within the framework of the Program “Universities of Russia”, Project No. UR.05.02.547, and with the financial support of the Wellcome Trust CRIG 069419 and the Russian Foundation for Basic Research Project 03-0448957. evident alternatives. As emphasized above, in most cases the compounds are stable enough and no reduction is needed. Only a small excess of the nucleophilic component is required during oxime or hydrazone synthesis, which makes the approach cost-effective. Side-reactions of O-alkyl hydroxylamines with traces of aldehydes and ketones in water are avoided by use of a small excess of aminooxy compound. Due to such considerations, the incorporation of an aldehyde group into the oligonucleotide chain seems to be the best route, because the molecules to be conjugated can normally be used in excess. Aminooxy oligonucleotides are more attractive for immobilization on aldehyde-coated surfaces, since the density of the array is higher compared to reductive amination. Aromatic hydrazines seem to be a good choice for hydrazone conjugation, since these form the most stable linkages in comparison to aliphatic hydrazines or hydrazides. However, in some cases a borohydride reduction is mandatory, e.g. when basic treatment is involved. Ketones are generally a poorer alternative to aldehydes. Although ketones are more stable to oxidants and milder reductants, and keto-derivatives of the oligonucleotides can be synthesized more easily, the lower reactivity of ketones makes their conjugation with amino compounds less practical. The possibility of conjugation with doublestranded nucleic acids could find broad application in the case of siRNA modification. In this review we highlighted the most significant contributions published to date in the field of application of addition-elimination reactions of a carbonyl group with nitrogen nucleophiles for nucleic acid chemistry. It is evident that there are now much more numerous
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