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Anych_en 71.83

Russian Chemical Reviews 71 (1) 71 ± 83 (2002) # 2002 Russian Academy of Sciences and Turpion Ltd Peptide nucleic acids: structure, properties, applications, strategies III. Applications of peptide nucleic acids IV. Basic principles of chemical synthesis of peptide nucleic acids V. Factors determining the efficiency of condensation in the synthesis of peptide nucleic acids VI. The main strategies of peptide nucleic acid synthesis VII. Some regularities of condensation reactions in the synthesis of peptide nucleic acids Abstract. The information on the structure and properties of peptide nucleic acids (PNA) is generalised. The use of PNA oligomers in biomolecular studies and biotechnology is exempli- fied. The published data on the most important methods for the chemical synthesis of PNA oligomers with the main emphasis on the efficiency of condensation reactions are considered. The methods for PNA synthesis are systematised; their advantages and disadvantages are discussed. Some recommendations for optimisation of the condensation procedure and synthesis of PNA are presented. The bibliography includes 153 references.
Peptide nucleic acids (PNA, 1) represent analogues of nucleic acids (NA, 2),1±4 but, in contrast to the latter, contain neither carbohydrate nor phosphate residues and have uncharged pseu- dopeptide backbones.1,5± 8 The monomeric unit of classical PNA comprises N-(2-aminoethyl)glycine and a heterocyclic base (purine or pyrimidine) bound through an acetyl linker. The monomers are linked by amide bonds. The geometry of the achiral backbone and its relative flexibility 3,9 confer on PNA an ability to mimic, with striking exactness, the spatial structure of carbohy- From the chemical standpoint, PNA represent a hybrid of an oligonucleotide wherefrom the nucleases have been adopted and a peptides, although neither the term `acid' nor `peptide' are peptide, the structure of which gave birth to the structural applicable to PNA, for in contrast to nucleic acids PNA are not principle of the PNA backbone. Thus, PNA possess properties polymeric acids and in contrast to peptides they do not contain of both these classes of compounds.8,10 This structural-and-func- amino acids. Nevertheless, the abbreviation `PNA' has now come tional duality of PNA determines their unique property.4 Indeed, into general use, although it would be more correct to refer to these molecules combine uniquely the strict recognising ability these compounds as polyamide analogues of oligonucleotides.10 inherent in NA with the flexibility and stability of proteins.
It should be noted that the term `peptide nucleic acids' is used to point to the structural similarity of these compounds to NA and to reflect the similarity of the PNA oligomeric backbone to that of Complementary PNA molecules form specific antiparallel PNA ± PNA duplexes having helical structures 4,11 similar to those of DNA and RNA duplexes but, which is even more S I Antsypovitch Department of Chemistry, M V Lomonosov Moscow important, they form highly stable specific (antiparallel and State University, Leninskie Gory, 119992 Moscow, Russian Federation.
parallel) duplexes with complementary DNA and RNA sequen- Fax (7-095) 939 31 81. Tel. (7-095) 939 31 48.
ces 1,5±7,9,12±14 containing Watson ± Crick base pairs.2,15,16 In all cases, antiparallel PNA ± DNA duplexes are more stable than the parallel ones, viz., their melting temperatures differ by *1 8C for each base pair.2 The circular dichroism spectra of PNA ± DNA Uspekhi Khimii 71 (1) 81 ± 96 (2002); translated by R L Birnova and DNA ± DNA duplexes are similar,2,17 which points to the formation of right-hand helices during the formation of PNA ± DNA duplexes despite the fact that base pairs in strand of PNA, while the second step includes rapid separation of PNA ± DNA and DNA ± DNA duplexes have slightly different geometries.17 NMR and X-ray crystallography studies of Peptide nucleic acids manifest high chemical and biological PNA ± PNA and PNA ± DNA complexes revealed that stabilities.25,46 They are highly resistant against cell nucleases, PNA ± PNA duplexes possess broad, deep major grooves and proteases and peptidases.25,27,47 PNA oligomers undergo very narrow, shallow minor grooves; noteworthy, one complete turn of slow enzymatic hydrolysis in both cell extracts and in vivo.25,46 a helix in PNA ± PNA duplexes corresponds to 18 base pairs, PNA molecules are distinguished by generally low toxicity and are whereas that in PNA ± DNA duplexes, to 13 base pairs.17±20 not prone to non-specific binding to cellular proteins. Being PNA ± PNA, PNA ± DNA and PNA ± RNA duplexes are immobilised on solid supports, PNA molecules preserve their considerably more stable than DNA ± DNA, DNA ± RNA and RNA ± RNA duplexes of the same compositions.1,2,11 ±17,21±23 It is known that modifications of NA backbones by replacing Even four-membered PNA sequences produce highly stable phosphodiester or carbohydrate units by non-charged or cationic duplexes with complementary DNAs.17 In contrast to NA ± NA structures may confer useful properties on NA, e.g., enhanced duplexes, the stabilities of PNA ± NA duplexes are little dependent resistance against nucleases and effective penetration through cell on the solution ionic strength.2,24± 27 PNA ± DNA duplexes are membranes and more specific and stronger binding to comple- formed faster than the corresponding DNA ± DNA duplexes,14,22 mentary target NA.48 Attempts are being made to improve the but high specificity of hybridisation is preserved.28 PNA structure in order to increase the ability of PNA for The stabilities of PNA ± DNA duplexes can be predicted based nonspecific binding to NA and their transport across cellular on a model which takes into account the interactions between only membranes.2,3,8 ±10,36 Thus the addition of certain peptides,49±53 the nearest adjacent bases;17,29,30 however, this model describes e.g., the 16-membered peptide `transportan',50,51 to PNA adequately the stabilities of short duplexes comprising no more increases considerably the rate of intracellular transport of PNA.9,23,49± 53 a-Helical PNA (aPNA) have been obtained in The most essential property of PNA is their unique sensitivity which the role of the backbones is played by a-helical peptide to mismatches in the structure of NA targets.7 The difference in structures.54±56 Such PNA analogues form highly specific stable melting temperatures of a perfect PNA ± DNA complex and a Watson ± Crick duplexes with complementary NA 54,55 and man- duplex containing one mismatch amounts to 20 8C and even ifest very high biological stabilities.56 PNA analogues with chiral backbones with positively and negatively charged groups, Peptide nucleic acids form PNA ± (DNA)2 triplexes with PNA ± DNA chimeras, etc., have been synthesised.36 double-stranded DNA.31±34 These triplexes are less stable than Comparative studies of structurally different PNA analogues the classical (DNA)3 triplexes.34 On the other hand, PNA forms have shown that classical PNA with N-(2-aminoethyl)glycine stable (PNA)2 ± DNA and (PNA)2 ± RNA triplexes with single- backbones first synthesised by Peter E Nielsen ten years ago 1 stranded DNA and RNA targets, respectively.5,12,14,16,33,35±39 manifest optimum NA-binding properties.4 Therefore, the main Such triplexes are often formed in the interaction of PNA with attention in this review will be given to methods of synthesis of double-stranded NA.37,40±44 In the latter case, the formation of classical PNA molecules based on N-(2-aminoethyl)glycine resi- triplexes leads to the displacement of one of the DNA strands resulting in complete separation or P-loop formation 38 with subsequent incorporation of PNA chains.5,33,37,40 ±44 PNA form III. Applications of peptide nucleic acids Watson ± Crick pairs with DNA. The attachment of the second PNA chain is accompanied by the formation of Hoogsteen By virtue of their unique properties,13,37 PNA have found wide pairs.35,39 It was found that Watson ± Crick chains of PNA are use in molecular-biological, biochemical, genetic engineering and antiparallel to DNA chains, while Hoogsteen strands are parallel clinical investigations.2,3,14,26,27,36,57 ±65 They represent attrac- tive candidates for new-generation genetic therapeutic drugs It is noteworthy that Hoogsteen strands stabilise Watson ± which interfere selectively with gene expression.9,14,26,36,57,62±71 Crick PNA ± DNA duplexes which are formed first; the latter can The use of PNA oligomers in antisense 9,26,27,36,57,67,69,72± 74 and antigen 27,36,57,67,68,72,75,76 biotechnologies is of considerable Homopyrimidine PNA molecules and PNA enriched with pyrimidine residues are especially prone to triplex forma- Antisense PNA selectively inhibit the expression of brain tion.9,35±39 The respective triplexes are extremely stable, e.g., proteins.47 The design of anticancer and antiviral drugs based on ten-membered (PNA)2 ± DNA triplexes have the melting temper- PNA seems to be a very promising approach.26,57,65 Some PNA atures of *70 8C.9,39 The ability of cytosine-containing PNA to derivatives manifest antibacterial 36 and antisense activities 9,47 form triplexes is pH-dependent, since cytosine can form Hoogs- towards eukaryotic cells and animal organisms.9,47 teen pairs with guanine residues only in the protonated state.9 No There is evidence that PNA interfere with all the key stages of triplexes with the composition (PNA)3 have been found.45 gene expression.9,46,57,77 Used in nanomolar concentrations, Recently, a new type of PNA-containing triplexes has been PNA cause a practically complete specific arrest of transcription discovered.37 Both PNA chains in the (PNA)2 ± DNA triplex of DNA templates.46,78 The usefulness of PNA oligomers in gene- formed upon binding of the PNA oligomer 5H-T4G2(TG)2-3H to oriented technologies has been demonstrated with a PNA- the oligonucleotide 5H-A4C2(AC)2-3H are in the antiparallel orien- dependent arrest of transcription elongation of RNA polymerase as an example.27,46,67,68,72 By virtue of their ability to induce The binding potential of PNA with respect to NA is far from effective blocking of transcription, PNA oligomers represent being exhausted. Studies of NA-binding properties of PNA aimed potential inhibitors of cell growth, which makes them a useful at the synthesis of novel PNA possessing improved structures and tool in the design of antitumour drugs.78 able to enhance the specific binding of PNA to complementary PNA duplexes and especially triplexes with mRNAs, e.g., PNA ± RNA and (PNA)2 ± RNA, effectively inhibit the trans- The melting of short-chain (PNA)2 ± DNA triplexes is a non- lation of mRNA.67,73,74 Their potent antisense effects in vitro equilibrium process, viz., the melting temperature depends on are due to high specificities and stabilities of triplex both the concentrations of components and heating velocity.38 (PNA)2 ± RNA.38 The arrest of translation elongation of mRNA Thus, the stabilities of (PNA)2 ± DNA complexes were found to be occurs even upon addition of six-membered complementary kinetic.38 The dissociation of such triplexes occurs in two steps, viz., the first (limiting) step includes separation of the Hoogsteen The antisense efficiencies of duplex-forming PNA are lower than those of triplex-forming ones; in this case, no less than 20- Peptide nucleic acids: structure, properties, applications, strategies and practice of chemical synthesis membered PNA are required for the inhibition of translation elongation of mRNA (however, the translation initiation can be arrested due to formation of even short-chain PNA ± RNA duplexes).73,74 Nevertheless, RNA molecules in hybrid PNA ± RNA duplexes are not cleaved by RNase H.67,73,79±81 The antisense effect of duplex-forming PNA is largely due to steric hindrances upon formation of stable PNA ± RNA complexes 9 which hinder the translation of mRNA. PNA-induced degrada- tion of mRNA, which is unrelated to the effect of RNase H, Duplex-forming PNA can inhibit translation in vitro, being synthesis of PNA are available; the most efficient of them have specifically directed against the binding sites of ribosomes, become especially popular in the past decade.14,94±97 whereas triplex-forming PNA are more specific against polypur- First of all, a solid-phase methodology is applied for the ine sites located `below' the translation initiation point.9 synthesis of PNA. The synthesis of oligomeric molecules on the Peptide nucleic acids are used for mapping of RNA molecules surface of polymeric supports was first developed by Merrifield in molecular biological studies, particularly for detection of RNA for the synthesis of peptides and proteins in 1962. The strikingly domains responsible for binding to other RNAs and peptides.80 simple idea to immobilise growing oligomeric chains on solid Their applications open up new possibilities for elaboration of supports has brought biooligomer synthesis to a qualitatively new novel approaches to the study of RNA ± RNA and RNA ± protein level. At present, the principle proposed by Merrifield 98 is widely interactions and such processes involving non-translatable RNA used in the synthesis of peptides and proteins as well as of DNA molecules as splicing. There is evidence that PNA molecules and RNA fragments (oligonucleotides). In the overwhelming behave as effective `traps' for some DNA-binding proteins.81 majority of cases, PNA oligomers are also synthesised on solid PNA ± DNA chimeras are convenient primers for DNA polymer- polymeric supports. In this review, the main emphasis will be laid ases.82 In recent years, PNA have extensively been used as on the problems related to the efficiency of solid-phase synthesis biomolecular tools for the studies of various intracellular proc- Although PNA oligomers can be synthesised by classical The use of PNA in the design of efficient procedures for the methods commonly employed in peptide synthesis,14 specially detection of hybridisation, which are extremely sensitive to mis- designed condensation procedures should be preferred, taking matches in NA targets, is a promising approach.28,83 Fluores- into account peculiarities of chemical structures of PNA mono- cently labelled PNA are used as diagnostic probes for detecting mers. In fact, of the different methods for the activation of specific NA sequences and for the study of penetration of PNA carboxy groups based on the use of activated esters, symmetrical oligomers through cellular membranes and their intracellular anhydrides, acid halides and in situ activating reagents, the in situ distribution.84±87 The use of PNA in combination with ion- activation has become the most promising approach, which is exchange HPLC (the detection limit is 150 pmol),88 MALDI TOF mass spectrometry,89 capillary electrophoresis 90 and other This method envisages the use of reagents based on uronium advanced analytical techniques 88 allows reliable identification of and phosphonium salts which effect fast (within several seconds) specific genetic sequences in various test samples.
activation of carboxy groups of PNA monomers. Owing to high The use of PNA in the design of electrochemical biosen- activation rates, mixing of PNA monomers with an activating sors 28,91±93 opens up new possibilities for fast screening of reagent can be performed directly in a column with a polymeric primary NA structures and helps overcome many problems of support to which the growing oligomeric chain is attached (it is this modern biotechnology.2 ±4,8,10,61 These compounds can be used procedure that represents in situ activation) or immediately before as an outstanding basis for the construction of new generations of the addition of the monomer to the reaction column. This makes it highly efficient diagnostic tools, e.g., biochips.83 possible to conduct PNA synthesis in an automated regime.
In this context, the development and optimisation of versatile The solid-phase procedure for PNA synthesis involving in situ techniques for the synthesis of PNA oligomers are becoming activation will be considered below; its advantages have been currently central tasks. The condensation of PNA monomers corroborated by chemical practice. The data on the efficiencies of and oligomers with formation of amide bonds induced by various other activation techniques can also be useful and proper consid- activating reagents is the key step in PNA synthesis. The combi- nation of protective groups, deprotection and capping conditions as well as post-synthetic work-up of synthetic PNA oligomers strongly depend on the condensation method used.
condensation in the synthesis of peptide nucleic IV. Basic principles of chemical synthesis of The yields of condensation products in the synthesis of PNA using The chemical synthesis of PNA molecules consists essentially in the in situ activation procedures depend critically on a number of the oligomerisation of the monomers 3 ± 6 comprising N-(2- factors. The most essential of them are as follows: aminoethyl)glycine backbones and acetic acid residues (acetyl Ð the nature and concentration of the activating reagent; linkers), each containing one of four nucleobases as a substitu- Ð the nature and concentration of the PNA monomer; ent.14 At present, a broad range of methods for the chemical Ð the nature of a nucleobase component of the PNA mono- mer and the nature of the nucleobase incorporated into the PNA Ð the nature and the concentration of a base (as a rule, Ð the presence or absence of catalysts, e.g., 1-hydroxybenzo- Ð the experimental procedure (e.g., preactivation of the PNA monomer or mixing of the PNA monomer with the condensation Ð condensation conditions (reaction time and temperature); conditions for PNA synthesis are compatible with those of peptide Ð other conditions (e.g., the quality of reagents, dryness of and oligonucleotide syntheses.104, 115 This opens up new oppor- solvents, inertness of the reaction atmosphere, etc.).
tunities for the synthesis of hybrid PNA ± DNA and PNA ± pep- It should be noted that information concerning the depend- ence of the yields of the condensation products on the nature of In addition to Boc and Fmoc protection, it was proposed to the heterocyclic bases of PNA monomers is practically absent.
use MMT groups for protection of 5H-terminal primary amino This problem demands special investigation. The majority of groups.96,107±112, 116 Although the MMT group, like the Boc literature sources cite average yields of PNA condensation prod- group, is acid-labile, this can be cleaved under considerably milder ucts calculated per coupling cycle of a hypothetical monomeric conditions than the Boc groups (the MMT groups are split off by fragment (irrespective of the nature of the monomer) or the total treatment with 3% trichloroacetic acid).107 In the MMT strategy, the amino groups of heterocyclic bases of the PNA monomers are It seems reasonable to consider all the factors mentioned usually protected by acyl groups (e.g., acetyl, isobutyryl, anisoyl, above. Hence, it is expedient to discuss in detail the main aspects benzoyl, tert-butylbenzoyl) (MMT/acyl version of the MMT related to the efficiencies of PNA condensations inherent in strategy).96,107, 110, 116 The reaction conditions are mild, which makes it possible to perform automated synthesis of PNA oligomers using the oligonucleotide synthesisers, while their VI. The main strategies of peptide nucleic acid compatibility with oligonucleotide synthesis protocols allows one to obtain PNA ± DNA chimeras.107± 109 The efficient condensation requires that the 5H-terminal amino Usually, PNA synthesis utilises conventional solid-phase peptide groups were not protonated, since in the form of cations they do synthesis protocols.14 Three synthetic strategies are currently not manifest nucleophilic properties. However, under basic con- especially popular which produce PNA in high yields and purity.
ditions where the amino group is not protonated and hence is These strategies differ in the nature of protective groups blocking active, the undesirable transfer of the N-terminal acetyl group 5H-terminal primary aliphatic amino groups in PNA monomers.
with the attached heterocyclic base to 5H-terminal primary ali- tert-Butoxycarbonyl (tBoc or Boc), 9-fluorenylmethoxycarbonyl phatic amino group may take place.94,97,115, 117 (Fmoc) and 4-methoxyphenyldiphenylmethyl (monomethoxytri- tyl, MMT) groups are generally used as protective groups, and Boc,12,94,99,100 Fmoc,77,97,101±106 and MMT strategies 96,107±112 of PNA synthesis are distinguished, correspondingly.
PNA Ð C-terminal fragment of the PNA oligomer.
The chemical nature of these groups and the differences in the This reaction can also occur under neutral conditions 94 conditions for their removal determine the choice of optimum resulting in the break of growing PNA chains and accumulation condensation reagents and condensation conditions for each of short-chain oligomers. This affords a mixture of products and particular strategy, although the main principles of PNA synthesis the isolation of target PNA presents a serious problem.
The formation of isomeric structures can be avoided provided The Boc strategy 12,94,99,100 was the first to be used for the the condensation is very fast. In this case, side products cannot be PNA synthesis. In this case, exocyclic amino groups of hetero- formed, since the N-acyl transfer is a rather slow proc- cyclic bases are usually protected by the benzyloxycarbonyl (Z, ess.94,97,100, 117 In the presence of reagents based on uronium and Cbz) group (the Boc/Z version),94 and O-benzyl groups are phosphonium salts, the condensation occurs so fast that even sometimes used as additional protective groups for guanine.113 in situ activation of carboxy groups of PNA monomers is possible.
The Boc/acyl version of the Boc strategy, where exocyclic amino Splitting of the N-terminal monomeric unit 12 under the action groups of nucleobases are protected by acyl groups, has also been of piperidine used for removal of Fmoc groups is yet another side described.95 One of the well-known disadvantages of the Boc strategy is the necessity to use strong acids for the removal of Boc groups (trifluoroacetic acid) and the cleavage of PNA oligomers from the polymeric supports (hydrofluoric acid, trifluoro- methanesulfonic acid, etc.). Such drastic conditions limit the range of PNA synthesised according to the Boc protocol.
A search for milder conditions has led to the development of the Fmoc strategy of PNA synthesis.14,77,96,97,101± 106,114 Here, the Fmoc groups are removed by mild treatment with piperi- dine.14 The benzyloxycarbonyl (Z),97 benzhydryloxycarbonyl (Bhoc) 106 or MMT groups 12,96,114 are used to protect amino groups of nucleobases. The advantage of a Fmoc/acyl version of this strategy 77,102±105 is the possibility of selective removal of the Fmoc groups without affecting the acyl protective groups of the heterocyclic bases.102, 103 The use of this strategy ensures higher yields of PNA in comparison with the Fmoc/Z strategy, while the Peptide nucleic acids: structure, properties, applications, strategies and practice of chemical synthesis It should be stressed that the problem of side reactions and the This was one of the first examples of the implementation of the efficiency of PNA synthesis on the whole depend critically on the Fmoc protocol to the synthesis of PNA. In this case, the use of the combination of protective groups used. Indeed, the Boc and Fmoc activated esters strategy ensured high efficiency of condensation.
strategies differ essentially in the conditions of deprotection of The yields of condensation products in the synthesis of PNA 5H-amino groups of the last added monomer. As mentioned above, oligomers with the chain lengths of up to 20 residues varied from the removal of Boc groups requires rather drastic acidic treatment, 95% to 99%.97 The average yields in the synthesis of PNA which leads to the protonation of 5H-amino groups. Therefore, this containing all the four types of nucleobases were 97% over each group should be neutralised before the addition of the next step, which corresponds to the 70% yield of target oligomers.
monomeric fragment, which is not required in the case of the With allowance for subsequent isolation, the yield of PNA On the other hand, the removal of Boc groups is always It is of note that the choice of strategies for the synthesis of quantitative and rather fast, whereas splitting of Fmoc groups by PNA is often determined by the necessity to obtain high yields of basic treatment proceeds slowly and not always completely,118,119 condensation products at low expenditures of expensive PNA which negatively affects the efficiency of condensation.
monomers where the use of manyfold (fourfold and higher) Aggregation of growing oligomeric chains is an additional monomer excess is undesirable. A combined use of the Fmoc obstacle to efficient condensation. Interchain aggregation makes protocol and PFP activation allows the use of as little as a twofold the deprotected terminal amino group only partly accessible for excess of PNA in a single condensation.97 The use of threefold (or subsequent condensation, which decreases the efficiency of the greater) excesses of PNA monomers did not result in further process on the whole.120, 121 It should be noted that aggregation is increase in the yields of condensation products. The dimethyl only possible in the case of the non-protonated amino sulfoxide (DMSO) ± N-methyl-2-pyrrolidinone (MP) mixture group.120,121 Apparently, repulsion of positively charged amino (1 : 4) appeared to be the solvent system of choice.97 Apparently, groups prevents the aggregation of oligomers. Thus, the inter- this system favours rapid access of the reagents to the growing chain aggregation never takes place in acidic media and terminal PNA oligomers.128 However, for other activation procedures, amino groups are more accessible to condensation. However, the other solvent systems were more efficient.
amino group cannot efficiently react with the activated monomer, The use of the activated esters approach to PNA synthesis since its nucleophilicity is suppressed.
sometimes gives reasonable results;12 however, in the majority of Deprotonation of terminal amino groups of PNA with simul- cases, higher (94% ± 99%) yields of condensation products were taneous condensation (neutralisation in situ) is the most elegant obtained using the in situ activating reagents.1,5 In principle, the approach to solving this problem. This methodology was first PNA synthesis can successfully be performed using the so-called employed for peptide synthesis in 1987.122 The use of neutralisa- carbodiimide activation involving, e.g., dicyclohexylcarbodiimide tion in situ in the synthesis of peptides using Boc and Fmoc (DCC) or N,N H-diisopropylcarbodiimide,12 which sometimes protocols results in a significant increase in the condensation affords high (up to 98% ± 99%) yields of condensation prod- rate.123± 125 This effect was especially spectacular in the synthesis ucts.6,7 Low rates of PNA condensation is the main disadvantage of `problematic' sequences which are especially prone to undergo interchain aggregation. Their syntheses by conventional methods Carbodiimide activation by DCC made it possible to obtain involving preliminary neutralisation of terminal amino groups addition products of thymidine and cytidine PNA monomers in quantitative yields. Purine monomers are only partly incorpo- At present, neutralisation in situ has become very popular for rated into PNA oligomers; repeated condensation does not result the synthesis of PNA along with conventional methods where in quantitative yields of the addition products.
deprotonation of 5H-amino groups of PNA oligomers precedes The use of N,N H-diisopropylcarbodiimide as the activating condensation. In some cases, the use of neutralisation in situ helps reagent has made it possible to obtain nearly quantitative yields solve the problem of side reactions of PNA isomerisation and even for purine monomers.99 However, this required the use of a increases the yields of condensation products.
fourfold excess of the monomers and the activating reagent, and the condensation lasted no less than 60 min.99 In addition, the VII. Some regularities of condensation reactions in introduction of the adenine and the guanine monomers into PNA oligomers required two and three condensation cycles, respec- A detailed knowledge of condensation reactions associated with The carbodiimide activation is a convenient procedure for PNA synthesis and a search for efficient procedures for its obtaining PNA adducts with other molecules. Thus the synthesis optimisation demand that the data available should be interpreted of hybrid PNA peptides containing biotin residues, which confer with due regard to the nature of reagents used for the activation of on PNA the ability to penetrate cell membranes efficiently, has PNA monomers. The methods for the synthesis of PNA ± DNA been described.129 The peptide fragment of the chimeric molecule chimeras are described separately, since in this case condensation was prepared using a standard peptide synthesis protocol,130 while the synthesis of the PNA fragment was carried out manually, using the Boc strategy based on the methods described in the 1. Activated esters and carbodiimide activation classical work by Nielsen.94 The activation with DCC was For the first time, the method of activated esters has been performed in the presence of 1-hydroxybenzotriazole, using a successfully employed for the synthesis of thymidine PNA oligo- fivefold excess of a PNA monomer. The condensation was mers.1,5 The synthesis of thymidine PNA using Boc-protected performed at an elevated (37 8C) temperature to increase the pentafluorophenyl (PFP) ester of a thymidine PNA monomer as a monomer was carried out in 1992.5 When the monomer concen- In other examples of the synthesis of hybrid PNA ± peptide tration was 0.1 mol litre71, the yield of the condensation product molecules,49 the Boc protocol was combined with carbodiimide was > 99%. However, an attempt to apply this method to the activation.94,129 Such an approach to the preparation of chimeras synthesis of cytidine PNA oligomers was without success: the yield is justified, since it ensures complete compatibility of syntheses of of the target product did not exceed 50% under identical con- both the peptide and PNA fragments of the hybrid molecules 49 and overall yields of target products of no less than 50%.49 There are some examples of successful applications of the A combined use of carbodiimide activation with the Boc/acyl method of activated esters for the synthesis of heterogeneous protocol common in peptide synthesis,130 allows one to obtain PNA. Thus the synthesis of PNA oligomers from Fmoc/Z- both classical PNA molecules with N-(2-aminoethyl)glycine back- protected monomers by ester activation has been described.97 bones and molecules with non-canonical backbones containing optically active monomeric fragments where the glycine residues heterocyclic bases. The use of the carbodiimide activation proce- are substituted by other amino acids, e.g., lysine, serine, isoleucine dure for the synthesis of such oligomers is usually less efficient.94 and glutamic acid.131 Incorporation of D-lysine-based monomers Studies of the effects of various factors, such as the nature of into PNA oligomers increases the stabilities of PNA ± DNA and activating reagents, solvents, monomer concentrations, the nature PNA ± RNA duplexes. With other amino acids, the stabilities of of the organic base (tertiary amine), catalysts, etc., on the these types of duplexes are usually low.131 efficiency of condensation in the in situ activation revealed that In some cases, such as in the synthesis of PNA ± peptide all of them are important for the optimisation of condensation chimeras and thymidine PNA oligomers, the activated esters and carbodiimide methodologies are employed. Sometimes, the yields Comparison of the efficiency of condensations under the of condensation products prepared by the activated esters (PFP) action of some uronium activating reagents, viz., the most popular method even exceed those obtained by in situ activation.97 reagents HBTU and TBTU and the relatively new reagents Nonetheless, it is generally acknowledged 12 that PNA syn- HATU and O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N H,N H-bis- thesis based on the use of in situ activation reagents, viz., uronium and phosphonium salts, is the most reliable approach, since it revealed that the condensation was efficient in all the cases under ensures higher yields of the condensation products. This approach study, but the yields of addition products were the highest with has considerably been developed in the recent years and it is this procedure that offers the broadest opportunities for the synthesis It should be noted that these results are valid exclusively for a DMF ± pyridine solvent system and for the concentrations of the PNA monomer and the base [diethyl(cyclohexyl)amine, DECHA] 2. In situ activation by reagents based on uronium and of 0.05 and 0.1 mol litre71, respectively. Under these conditions, the average yields of the condensation products were The most popular activating reagents for PNA synthesis are 92.2% ± 97.1% irrespective of the nature of PNA monomers.
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium However, conditions can be selected where other activating phosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetra- reagents will be more efficient than HBTU, e.g., in other solvent methyluronium hexafluorophosphate (HATU), (benzotriazol-1- systems or in the presence of other bases. This suggests that condensation conditions are to be chosen for each activating O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-[(ethoxycarbonyl)cyanomethyl- Comparison of the efficiencies of HBTU and PyBOP has demonstrated the former to be superior under identical condi- (TOTU) and (benzotriazol-1-yloxy)-tris(dimethylamino)phos- tions. The overall yields of PNA oligomers using HBTU and phonium hexafluorophosphate (BOP), although other reagents PyBOP activation were 61% and 40%, respectively, which corre- sponds to average yields of 97.1% and 94.7% in each step.94,100 It is of note that this comparative study was carried out under An important role in efficient condensation is played by the solvent system used. Thus in a DMF ± pyridine mixture, the overall yield of the condensation product was 61%, whereas those in DMF or DMF ± DMSO were 55% and 22%, respec- tively.94 However, it was not indicated which monomers deter- mined the lowest yields of PNA. Noteworthy, virtually none of the works cited in this review provide these data.
Studies of the dependence of the yield of a PNA oligomer on the nature of an organic base (tertiary amine) have shown that 4-dimethylaminopyridine (DMAP), DECHA, (dicyclohexyl)me- thylamine and (dicyclohexyl) ethylamine were as efficient as (diisopropyl)ethylamine (DIPEA) which is widely used in peptide The nature of the tertiary amine only slightly affected the yields of the condensation product (93.7% ± 95.3%). The con- densation in the presence of DIPEA in the DMF ± pyridine system is not optimum, since this amine reacts with the monomers to give insoluble salts. Better conditions for this reaction can be found.
The condensation in the presence of DIPEA is efficient in Studies of relationships between the yields of condensation products and concentrations of PNA monomers revealed that acceptable yields can be obtained when the monomers are used at concentrations no less than 0.1 mol litre71. At lower concentra- Since condensation strongly depends on the strategy used for tions (0.05 mol litre71), the condensation proceeds as a rule too PNA synthesis, in the first place, on the combination of protective slowly resulting in the accumulation of side products from groups, it seems expedient to classify the data on the in situ activation into three groups corresponding to Boc, Fmoc and Addition of catalytic amounts of DMAP and 1-hydr- oxybenzotriazole to the reaction mixture may have a negative effect on the efficiency of condensation, although it is known that their addition sometimes favours the formation of amide bonds.94 The Boc/Z-modification of the Boc strategy is a classical approach The loading { of the polymeric support should not exceed to PNA synthesis. The dependence of the condensation efficiency 0.1 ± 0.2 mol-equiv. g71, which is essential for the maximum yield of the Boc/Z strategy on different factors has been studied in sufficiently great detail.94 This strategy makes it possible to obtain { Here, loading is expressed as the number of functional groups per unit high yields of PNA containing > 15 units with all the four types of Peptide nucleic acids: structure, properties, applications, strategies and practice of chemical synthesis of the PNA oligomer. With a higher degree of loading, the previously developed procedures,94,131,132 the Boc strategy is efficiency of PNA synthesis is lower.94,100 combined with HBTU activation in the presence of DECHA.
The use of the Boc/Z strategy ensures high (99.4%) yields of This approach affords high yields of condensation products, but target products with in situ activation (HATU) in the presence of requires the use of a fourfold excess of PNA monomers.79 DIPEA.95 In this case, the reaction mixture must contain a large The Boc strategy is also used in the synthesis of PNA (e.g., sevenfold) excess of the PNA monomer with respect to oligomers containing modified units.131, 133±135 Thus PNA mole- loading of the polymeric carrier.95 The amount of the activating cules may incorporate units containing anthraquinone and acri- reagent is usually reduced by 10%, while the tertiary amine is dine residues.133 Such oligomers are used to study the melting taken in a twofold excess with respect to the PNA monomer.88 behaviour of hairpin-shaped PNA of high-molecular-mass Acceptable yields are obtained with a fourfold excess of the PNA ± PNA duplexes by fluorescence quenching (the so-called PNA monomer and by activation with HATU (the amount of `molecular beacon' method). High yields of condensation prod- HATU is 0.9 mol-equiv. with respect to a monomer) in the ucts are obtained. Its repeated condensation is used for the presence of DIPEA and lutidine.47 The use of a fivefold excess of attachment of a modified PNA monomer followed by a non- PNA monomers and a tenfold excess of DIPEA makes it possible to increase the overall yields of PNA oligomers to 92%.17 The Boc/Z method combined with HBTU activation 94,134 Very often, the yields can be increased upon preactivation of allows synthesis of PNA molecules with fluorescent labels at their PNA monomers. To this end, the PNA monomer is mixed and N-termini.134 Such PNA derivatives can be used for detecting incubated with the activating reagent for several seconds before specific NA sequences. The fluorescence of the label may increase being loaded onto the column.95,96,109,110, 113,115, 116 Strictly more than 50-fold upon hybridisation of PNA with the comple- speaking, here we do not deal with the in situ activation, however, mentary NA target in comparison with that of the free PNA.134 the term `in situ activation' is conventionally related to the nature The classical Boc/Z strategy is also used for the synthesis of of the activating reagent rather than to the order of mixing of the PNA chimeras containing clusters of modified fragments, e.g., reagents. It is of note that the methodology of preactivation does those with positively charged, chiral backbones built up of not imply the isolation of activated monomers. Usually, the D-lysine residues.135 The presence of such fragments in PNA amount of the activating reagent is 5% ± 10% smaller than that confers useful properties on the latter.131, 135 In contrast with ordinary PNA, chimeric PNA form exclusively antiparallel Preactivation of PNA monomers by incubating them with the duplexes with DNA; their stability depends critically on the activating reagent for 2 min and condensation in an MP ± pyr- presence of mismatches. Even one mismatch decreases sharply idine mixture make it possible to obtain PNA oligomers in overall the stability of hybrid duplexes. If a mismatch is located in the yields of 90%. Depending on the length and composition of middle of clusters containing three modified residues, ten-mem- sequences, the yields of PNA vary from 68% to 90%.88 bered PNA ± DNA duplexes are unstable even at 15 8C In a search for the most rational technique for PNA synthesis, (DTm = 28 8C). Such PNA can serve as the basis for the develop- an investigator has to select between a rapid and cheap synthesis ment of genetic diagnostic tools, particularly, for the detection of with the use of a small excess of PNA monomers and low (but acceptable) yields and lengthy syntheses with large expenditures of Synthesis of these PNA derivatives by the Boc strategy expensive monomers and activating reagents, but giving nearly combined with HBTU activation in the presence of DECHA,131, 136 affords chiral oligomers in overall yields of Attempts to specify the conditions for effective synthesis of 80% ± 90%,135 and optical purity of no less than 90%.
PNA have been undertaken time and again.95 Primary attention in In the majority of cases, the Boc protocol includes the use of the optimisation of PNA synthesis is usually given to such factors standard polymeric supports containing (4-methylbenzhydryl)- as the low cost and ease of synthesis of PNA monomers, nearly quantitative yields of condensation products, the simplicity and efficiency of the procedure for isolation of PNA oligomers from the reaction mixture after completion of the synthesis and the possibility to obtain chimeric DNA ± PNA duplexes and ligand- Such a choice of optimisation parameters seems to be justified, however, other factors, such as reaction rate and economy, are no less important. Thus the rate of condensation should be high enough to minimise side reactions. The synthetic strategy should first of all be efficient and allow for economic expenditure of PNA oligomers are cleaved from the solid phase by treatment expensive PNA monomers and activating reagents. The latter factor is of crucial importance in large-scale syntheses of PNA.
The Boc strategy allows the use of various activating reagents While comparing the Boc/Z and the Boc/acyl strategies of including BOP.137 The use of BOP and DIPEA as a base affords PNA synthesis, preference is given to the latter, since it is thought high yields of condensation products in various solvent systems, to be more promising. Although the average yields of oligomers e.g., DMF ± CH2Cl2 and DMF ± DMSO. In this case, the use of a calculated per one condensation cycle are high in both cases, viz., twofold excess of PNA monomers is sufficient, but each conden- no less than 98% (Boc/acyl) and about 99% (Boc/Z), the yields of sation reaction should be repeated in order to provide more PNA oligomers in the case of the Boc/acyl synthetic strategy and conventional isolation procedure do not exceed 65% (relative PNA can also be synthesised using the Boc/Z strategy com- to the support loading), and are as low as 20% according to the bined with the in situ activation with TBTU in DMF ± pyridine 113 Boc/Z protocol.95 In addition, the Boc/acyl strategy allows one to as described in the classical work of Nielsen.94 However, it is more synthesise PNA ± DNA chimeras and to prepare addition prod- expedient to carry out the condensation in DMF in the presence of ucts of acid-labile ligands to PNA, which is inattainable in the case a twofold excess of DECHA as a base with respect to the monomer. This is associated with the good solubility of PNA The synthesis of thymidine PNA oligomers makes use exclu- monomer salts formed in DMF.113 The use of a threefold excess of sively of Boc-protection of 5 H-terminal amino groups, since the PNA monomers is optimum. This approach allows one to use thymidine PNA monomer does not require protection of the 2-amino-6-benzyloxypurine with the non-protected amino group heterocyclic base.77,79 Such oligomers are conveniently synthes- as a guanine precursor.113 The side reaction (capping) of the ised by manual techniques.79 In this case, by analogy with oligomeric chain can be avoided if the amount of TBTU is 10% less than that of the PNA monomer 113 or if the PNA monomer is It is generally recognised that the Fmoc strategy of PNA syn- Capping of oligomeric chains occurs both in PNA and peptide thesis14,77,96,100±106, 114 requires milder conditions than the Boc synthesis 123 provided the free activating reagent is present in the strategy.12 In particular, no treatment of PNA oligomers with reaction mixture. This is possible owing to the fact that the strong acids before and after synthesis is necessary. This allows the terminal amino group of the peptide following deprotection reacts use of other protective groups for exocyclic amino groups of with both the activated amino acid derivative and the activating heterocyclic bases of the PNA monomers. Correspondingly, the reagent.123 Thus in the presence of an excess of HBTU, the Fmoc strategy affords higher degrees of purity of reaction tetramethylguanidine derivative was formed, which did not mixtures and higher overall yields of PNA oligomers. The Fmoc/ undergo subsequent elongation of the peptide chain.138 This side acyl strategy of PNA synthesis is especially promising,77,104, 105 reaction can be avoided if the amount of the activating reagent is since it can also be used for the synthesis of PNA ± DNA and smaller (by 5% ± 20%) with respect to the monomer.113 With TBTU as the activating reagent, another side reaction, Various versions of the Fmoc strategy are currently known.
viz., the N-acyl transfer, can take place.94,113, 123 To avoid this, Thus Bhoc protection of heterocyclic bases of the PNA monomers neutralisation in situ is used.123 In this case, condensation is and HATU activation allows automated synthesis on oligonu- carried out in the presence of a base without preliminary neutral- cleotide synthesisers.106 The use of a DIPEA ± lutidine mixture isation of the 5 H-terminal primary amino group of the oligomer.113 seems to be more effective than the use of only one base, since it The efficiency of condensation is monitored by HPLC analysis of affords higher yields of the condensation products.106 aliquots obtained by appropriate treatment of a small portion of a DMF is a suitable solvent for the Fmoc condensation, and polymeric carrier (3 ± 5 mg) following attachment of the next HATU is one of the most potent activating reagents.141 However, in this case, too, the synthesis of individual PNA oligomers may In the synthesis of thymidine PNA oligomers based on the use face problems related to non-efficient condensation.141 Thus the of the Boc strategy and TBTU activation, a decrease in the yields synthesis of PNA oligomers with sequences containing several of condensation products is observed sometimes after addition of identical consecutive heterocyclic bases (not necessarily purines) the first 3 ± 4 monomeric fragments.113 This leads to the accumu- yields short-chain products. This problem can partly be overcome lation of short chains, whereas the overall yield of PNA does not through the use of repeated condensations.141 exceed 30% (in the case of a 10-membered oligomer). The same Two condensation cycles are carried out in the following cases: problem sometimes arises with HATU activation, presumably, in the synthesis of sequences containing four or more identical due to aggregation of growing thymidine PNA oligomers. This consecutive residues after attachment of two or three identical can partly be overcome through the attachment of lysine residues PNA monomers to the oligomer, in the synthesis of PNA at the C-ends of PNA chains, which prevents the interchain oligomers containing purine clusters and in the synthesis of aggregation of the oligomers formed.
18-membered and more extended PNA oligomers independent It is noteworthy that the synthesis of heterogeneous PNA of the composition of the sequence after the attachment of the oligomers containing monomeric fragments of all the four types is not accompanied by significant reduction of product yields in the The yields of PNA obtained using the Fmoc strategy and PNA synthesis.113 In this case, the yield over each condensation HATU activation vary from 26% to 38%.141 In some cases, the step reaches 97%, which corresponds to 66% overall yield of the overall yields of PNA oligomers do not depend on the sequence lengths but are determined by the efficiency of the attachment of Similar problems, viz., aggregation of PNA chains, may arise the first PNA monomer to the polymeric support, since the yield during the removal of protective Z groups after completion of of the first condensation product can be lower than the yields of PNA synthesis resulting in a significant decrease in the yields of the products obtained in subsequent condensations.141 PNA.11,113 A crucial role in this process belongs to complemen- In the majority of cases, reversed phase HPLC is used for the tary interchain and intramolecular coupling of PNA oligomers.
analysis of reaction mixtures and isolation of target products Even four-membered PNA ± PNA duplexes significantly hinder under conditions for the separation of peptides rather than post-synthetic work-up of PNA oligomers.113 Therefore, oligo- oligonucleotides.141 The condensation efficiency of the Fmoc meric PNA sequences should be analysed for the possibility of strategy can also be estimated spectrophotometrically by measur- intramolecular hairpin and intermolecular cluster formation prior ing UV absorption of a product formed upon removal of Fmoc to the synthesis. It was found that the overall yields of PNA can be groups by piperidine in the range of 256 ± 301 nm. This method is increased to 75% and even more through incorporation of a lysine especially convenient for stepwise monitoring of PNA oligomer- residue into PNA heterooligomer, since the charged e-amino group of lysine partly prevents the aggregation of PNA chains.
Thus, the Fmoc strategy can be used for the synthesis of PNA These data suggest that the efficiency of synthesis of PNA oligomers unattainable by other methods. This procedure allows oligomers strongly depends on the properties of PNA sequences to modifications to the synthetic protocol aimed at increasing the be prepared irrespective of the synthetic procedure used.139 Theo- yields of condensation products in separate synthetic cycles.141 retically, PNA may contain any combination of monomers, while The low efficiency of the condensation encountered in the the synthesis of certain sequences in quantitative yields faces Fmoc strategy of PNA synthesis seems to have the same reasons difficulties. Thus the attachment of a purine monomer to an and solutions as those in peptide synthesis.142 It is known that oligomeric PNA sequence containing a 5 H-terminal purine base is many problems of automated solid-phase peptide synthesis are problematic. Therefore, if PNA containing purine clusters are to be related to the nature of the sequence to be synthesised.142 Low synthesised, the synthetic protocol should include repeated con- condensation yields are due to the formation of bulky spatial densations.139 The lowest yields were obtained in the synthesis of peptide structures, which may interfere with the formation of PNA oligomers containing several consecutive guanine residues.
In conclusion, it may be said that the Boc strategy can be Those peptide chain fragments which are susceptible to implemented in both manual and automated variants.139 interchain aggregation, can also decrease the accessibility of the Although the former seems to be rather efficient and inexpen- amino group and thus prevent further elongation of the chain.120 sive,140 automated synthesis using peptide and oligonucleotide Spatial hindrances appear in the course of PNA synthesis synthesisers holds especially great promise.141 The reason is that which decelerate the removal of protective Fmoc groups from the the manual synthesis of PNA is not only lengthy and laborious, 5 H-termini of PNA oligomers and decrease the efficiency of but also needs large-scale synthesis (not less than 5 mmol) in order to obtain acceptable yields of condensation products.
Peptide nucleic acids: structure, properties, applications, strategies and practice of chemical synthesis The synthesis of purine-rich PNA presents a special problem.
monomeric unit to a polymeric support have also been If a PNA sequence contains more than two consecutive purine residues, the efficient attachment of the next purine monomer The best conditions for the PNA synthesis by the Fmoc/acyl requires a longer reaction time and repetition of the condensation strategy include the use of a twofold monomer excess procedure three or four times. The attachment of the guanine (0.125 mol litre71), preactivation with HATU (0.8 mol-equiv.), monomer to the 5 H-terminal guanine unit proceeds at a very slow DIPEA and lutidine (1 ± 2 min), condensation (20 min) and repetition of the condensation procedure in each (with the Aggregation of PNA chains is the main reason for low exception of the first) reaction cycle. This provides an overall condensation efficiency inherent in both Fmoc and Boc strategies.
yield of heterogeneous PNA of 43% (for 16-membered oligom- The synthesis of partly or fully self-complementary sequences may ers), which corresponds to the average yield of the condensation produce problems relevant to intrachain association of PNA product of 95% (in this case, the attachment of the first mono- oligomers. In addition, the solid-phase synthesis conditions meric fragment occurs with high yield). The main disadvantage of favour intermolecular aggregation of growing oligomers. Thus this method is the necessity to repeat the condensation procedure purine-rich PNA and thymidine PNA oligomers (both four- in each cycle.115 The expedience of this approach is doubtful membered and more extended ones) are prone to aggregation.
despite its obvious advantages, viz., good reproducibility of The condensation efficiency strongly depends on such factors as the loading of the polymeric support and the composition of the solvents. Thus, low densities of oligomers `growing' on solid supports favour solution of the problem of intermolecular aggre- This strategy of PNA synthesis is rather promis- gation of PNA chains, whereas certain solvents prevent inter- and ing 38,96,107± 112,116, 144,145 particularly its MMT/acyl ver- intramolecular aggregation of PNA molecules (e.g., the formation sion,96,107, 110,116 which is fully compatible with oligonucleotide of hairpin structures) and ensure effective diffusion of reagents to synthesis protocols. It allows the use of automatic DNA synthes- the N-termini of the growing PNA chains.
isers without modification of their design or software, which is Studies aimed at optimisation of conditions for PNA synthesis especially convenient for conducting the syntheses of PNA ± DNA are currently under way, since no ideal method for PNA synthesis has been developed so far. The Fmoc/acyl strategy of PNA The MMT strategy allows the use of a broad range of synthesis seems to be the most advanced one, since it permits one activating reagents,38,112, 144,145 including mesitylenesulfonyloxy- to obtain oligomers of virtually any composition in high benzotriazole (TMSOBt) 112 and 3,4,6-triisopropylbenzenesulfo- nyloxybenzotriazole (TPSOBt).144 The latter gives higher yields of Thus the synthesis of thymidine PNA oligomers by the Fmoc/ the condensation products (the average yield in one cycle reaches acyl method combined with HATU activation requires only small 96%) than the commercially available activating reagent (twofold) excess of the monomer 115 and a 20% deficiency of the PyBOP.112, 144 Depending on the nature of the PNA monomer activating reagent. It is more expedient to use MP as a solvent and and the 5 H-terminal oligomeric fragment, the yield in each the DIPEA ± lutidine mixture as a base. Preactivation of PNA condensation step varies from 91% to 99%.144 monomers for 2 min is yet beneficial. The yield of the condensa- The efficiency of condensation carried out by the MMT tion product in each step is 85% ± 90%, the condensation time is strategy can be estimated spectrophotometrically by measuring 30 min and the overall yields of the 7-membered oligomer is no UV absorption spectra of the MMT+ cation formed upon less than 50%.115 The efficiency of the reaction is most conven- removal of the protective MMT group from the 5 H-terminal iently monitored spectrophotometrically.
amino group of a PNA oligomer. The MMT strategy combined Attempts have been made to optimise condensation condi- with TPSOBt activation is compatible with oligonucleotide syn- tions in solution using mixtures of PNA monomers and amino thesisers.112 This approach was first used in the synthesis of PNA acid esters as model systems.115 Direct application of the data molecules containing uracil residues.112 The `manual' variant of obtained in these model systems to solid-phase PNA synthesis is the MMT strategy is also effective, e.g., in the synthesis of questionable, since the optimum conditions for the synthesis in solution and on polymeric supports may differ in principle. This The use of PyBOP as an activating reagent allows one to reach circumstance should be taken into consideration when selecting 95% ± 99% yields in each step of the monomeric fragment model systems. On the other hand, these studies sometimes give coupling.96 However, in this case, the use of concentrated (0.3 M) solutions of PNA monomers and PyBOP is necessary. It The yields of condensation products in the reaction of PNA is more expedient to use N-ethylmorpholine as a base and to monomers with L-phenylalanine tert-butyl ester were not lower than 95% irrespective of the nature of the activating reagent The MMT strategy is used for the synthesis of phosphonate (HATU or HBTU in the presence of 1-hydroxybenzotriazole), of analogues of PNA (pPNA).110, 116,146, 147 The presence of nega- the tertiary amine (N-methylmorpholine, lutidine or the tively charged groups in the pPNA backbone makes PNA DIPEA ± lutidine mixture) and of the solvent (DMF or MP).
analogues readily soluble in aqueous solutions in comparison The nature of the base and the activating reagent did not influence with classical PNA oligomers. These molecules bind specifically to the efficiency of the reaction. The best result was obtained in the complementary fragments in DNA and RNA, although the case of HATU activation in the presence of lutidine in DMF.115 melting temperatures of pPNA ± NA complexes are somewhat As mentioned above, the overall yields of PNA oligomers may lower than those of the corresponding PNA ± NA complexes.
depend on the efficiency of attachment of the first monomer to the A combination of the MMT/acyl protocol with triisopropyl- polymeric support. Thus the yield of the attachment product of benzenesulfonylnitrotriazole activation is efficient in the synthesis the first cytosine monomer to the polymeric support (Tentagel) in of pPNA.110, 116,146, 147 The condensation in the presence of the synthesis of heterogeneous PNA by the Fmoc/acyl strategy N-methylimidazole as a nucleophilic catalyst permits one to combined with conventional HBTU activation in the presence of obtain the average yields of 95% in the condensation step with 1-hydroxybenzotriazole and lutidine did not exceed 50%,115 a reaction time of 10 min.110, 116 However, the quantitative over- whereas those obtained in subsequent condensation steps were all yields of condensation products are not achieved, although the no less than 80%. The efficiency of attachment of the first PNA use of dilute solutions of PNA monomers (0.05 M) and the monomer can be increased to 80% and even higher using repeated activating reagent (0.06 M) together with preactivation (mixing condensation. In this case, the time for each condensation can be of the PNA monomer with the activating reagent and N-methyl- reduced.115 Low yields of the attachment products of the first imidazole), makes this procedure attractive.110, 116 3. Some peculiarities of the synthesis of PNA ± DNA The synthesis of PNA fragments of such chimeric oligomers usually employs the MMT/acyl strategy. In this case, the con- The interest in PNA ± DNA chimeras has arisen in the past ditions of PNA synthesis are compatible with those of oligonu- decade, which gave a strong impetus to the development of cleotide synthesis;36,149 PNA fragments can be synthesised by methods for their synthesis.77,109, 111,144, 148± 150 The use of manual techniques. The condensation is performed in the classical PNA in biochemical studies is limited due to their poor DMF ± pyridine mixture with 2-[2-oxo-1(2H)-pyridyl]-1,1,3,3- solubilities in aqueous solutions, proneness to self-aggrega- bis(pentamethylene)uronium tetrafluoroborate (TOPPipU) as tion 4,12,34 and low penetrability through cell mem- the activating reagent and DECHA as the base.153 Under these branes.4,9,84,151 The latter is the main obstacle for the use of conditions, the condensation of thymidine and cytidine PNA canonical PNA oligomers as antisense agents in vivo.9 monomers proceeds smoothly, purine monomers are attached Chimeric PNA ± DNA molecules are devoid of most of these inefficiently to the growing PNA chain.149 If HATU is used as the drawbacks. They possess all the advantages of PNA together with activating reagent, DIPEA as the base and acetonitrile as the valuable properties inherent in NA. Indeed, the PNA ± DNA solvent in the presence of a fivefold excess of PNA monomers chimeras synthesised so far combine high biological stabilities, (necessary to attain high yields of condensation products), the high affinities and selectivities of binding to NA targets typical of reaction time is no less than 15 min.34 This method was used for PNA with perfect solubilities and the ability to activate hydrolysis the synthesis of chimeric molecules containing 5-bromouracil and of RNA targets by RNAse H, which are characteristic of 5-methylcytosine residues.34 The incorporation of 5-methylcyto- DNA.9,34 Chimeric PNA ± NA molecules have various sine residues into the PNA chains of chimeric molecules increases applications, viz., they are promising therapeutic (including the stabilities of their duplexes and triplexes with complementary antisense) drugs 9 and can be used as a basis for highly effective The modified thymidine PNA monomer based on N-(2- probes.77,96,107±109, 144, 148,149 Synthesis of hybrid PNA ± NA hydroxyethyl)glycine 34,107, 148 is often used as a linker between molecules manifests specific features, which necessitates a consid- DNA and PNA fragments of chimeric molecules of the 5 H-DNA ± PNA-3 H type; the latter can be synthesised using both The correct choice of linkers between PNA and DNA frag- Boc 148 and MMT strategies.34,107 If the Boc/Z strategy is used, ments of the chimeric molecules is one of the most important the PNA synthesis is carried out on a solid phase; the linkers are problems. Depending on whether the 5 H-terminal fragment of the attached under the same conditions.148 Subsequent synthesis of hybrid molecule belongs to PNA or DNA, the linker used is DNA fragments of hybrid molecules should also be performed on represented either by modified nucleosides (e.g., 5 H-amino-2 H,5 H- the solid phase. It is inadmissible to perform the synthesis of DNA dideoxynucleosides 77,149) or modified PNA monomers [e.g., fragments in solution, since the solubility of PNA oligomers N-(2-hydroxyethyl)glycine derivatives].148 devoid of protective groups in organic solvents is insufficient to provide efficient condensation with phosphoroamidite derivatives The solid-phase MMT method is more suitable for the syn- thesis of the 3 H-PNA fragments; after completion of PNA syn- thesis, solid-phase synthesis of the DNA fragment is continued without detachment of the oligomer from the support. This prevents the use of PNA monomers the heterocyclic bases of Syntheses of both types of PNA ± DNA hybrids, viz., which are protected by acid-labile groups, since the DNA frag- 5 H-PNA ± DNA-3 H and 5 H-DNA ± PNA-3 H, have been described.
ments will not withstand acid treatment used to remove protective Owing to the charged backbones of their DNA fragments, groups. This synthetic procedure is inapplicable to chimeric PNA ± DNA chimeras are perfectly soluble in aqueous solutions, molecules containing PNA monomers of all the four types, but which makes possible their isolation and analysis by standard can be used for the synthesis of hybrid molecules with the methods, such as polyacrylamide gel electrophoresis and ion- pyrimidine monomers constituting the PNA fragments.148 The MMT/acyl modification of this method is devoid of these In the case of 5 H-terminal PNA fragments, PNA and DNA disadvantages and allows the synthesis of PNA ± DNA hybrids fragments are linked by the amide bond and 5 H-amino-2 H,5 H- containing all the four types of nucleobases in both DNA and dideoxynucleosides are used as linkers.77 Such hybrid molecules are synthesised by various methods. Thus 5 H-PNA ± DNA-3 H The MMT/acyl strategy allows the application of the fully hybrids are prepared according to Boc/Z protocols commonly automated protocol on oligonucleotide synthesisers. The DNA used in the synthesis of PNA-peptide conjugates.152 However, the fragments of hybrid molecules are usually synthesised according use of this technique for the synthesis of PNA ± DNA chimeras to a conventional phosphoroamidite protocol, which makes use of containing purine nucleotide residues may result in acid-catalysed commercial 2 H-deoxynucleoside phosphoroamidites.108 This apurinisation of the DNA fragment during deprotection of method of synthesis of PNA ± DNA chimeras has practically no heterocyclic bases of PNA.77 Therefore, this approach is used exclusively for the synthesis of PNA ± DNA hybrids the DNA The synthesis of PNA fragments of 5 H-PNA ± DNA-3 H fragments of which contain more stable pyrimidine nucleotides, oligomers may involve HBTU activation in the presence of whereas 5 H-PNA ± DNA-3 H chimeras are more efficiently synthes- DIPEA in DMF ± acetonitrile.150 Solutions of PNA monomers and activating reagents should be used at concentrations of no less If hybrid molecules contain 5 H-terminal DNA fragments, the than 0.1 M (preferably, 0.2 M); preactivation is also desirable. A PNA and DNA parts can be linked by phosphoramide bonds commercially available aminohexanol phosphoroamidite deriva- without any linkers. In this case, the synthesis of PNA fragments is tive can be used as a linker between the PNA and DNA fragments; carried out using the Fmoc/acyl protocol. With the thymidine this is attached to the 5 H-end of a DNA fragment by a standard monomer, Boc-protection of the 5 H-terminal amino group is oligonucleotide synthesis protocol. Then the synthesis of a 5 H-ter- possible. The activation is performed with HATU in the presence minal PNA fragment of a chimeric molecule is followed.150 of DIPEA and DMAP; the condensation is carried out in DMF.
The MMT/acyl strategy is used in the synthesis of chimeric In this case, the use of acid-labile groups for protection of molecules with the composition 5 H-PNA ± DNA ± PNA-3 H.109 In heterocyclic bases of PNA monomers is inadmissible because of this case, both PNA fragments are synthesised in an automated easy acid hydrolysis of phosphoramide bonds.77 regime using HBTU activation in the presence of DIPEA in DMF ± acetonitrile mixture; this may require an eightfold excess Peptide nucleic acids: structure, properties, applications, strategies and practice of chemical synthesis of the reagents with respect to the carrier loading. Preactivation of 15. SC Brown, SA Thompson, J M Veal, D G Davis Science 265 777 PNA monomers makes it possible to increase the condensation efficiency and the average yields in the attachment of monomeric 16. M Eriksson, P E Nielsen Q. Rev. Biophys. 29 369 (1996) 17. N Sugimoto, N Satoh, K Yasuda, S-I Nakano Biochemistry 40 8444 Obviously, the problem of efficiency of each individual approach to the synthesis of PNA molecules has no unambiguous 18. M Leijon, A Graslund, P E Nielsen, O Buchardt, B Norden, solution. The choice of the most adequate strategy for the PNA SM Kristensen, M Eriksson Biochemistry 33 9820 (1994) synthesis depends on the goal and facilities as well as on the 19. M Eriksson, P E Nielsen Nat. Struct. Biol. 3 410 (1996) number of PNA oligomers to be synthesised, the scale of synthesis, 20. R Rasmussen, J SKastrup, J N Nielsen, J M Nielsen, P E Nielsen composition and purity of PNA oligomers.
21. C Meier, J Engels Angew. Chem., Int. Ed. Engl. 31 1008 (1992) 22. D J Rose J. Anal. Chem. 65 3545 (1993) 23. W M Pardridge, R J Boado, Y-SKang Proc. Natl. Acad. Sci. USA The design of the most efficient method for the synthesis of PNA 24. J C Norton, M A Piatyszek, W E Wright, J W Shay, D R Corey oligomers requires that a rational compromise between the efficiency and economy of the synthetic process be found.
25. V V Demidov, V N Potaman, M D Frank-Kamenetskii, On the one hand, one has to reach the maximum yields of M Egholm, O Buchardt, SH Sonnichsen, P E Nielsen Biochem.
condensation products and the choice of synthetic strategy must take into account both the nature of the activating reagent and 26. L Good, P E Nielsen Antisense Nucl. Acids Drug Devel. 7 431 (1997) other factors discussed in this review.
27. P E Nielsen, M Egholm, R H Berg, O Buchardt Anti-Cancer Drug On the other hand, the synthesis of PNA should be rational.
This implies that the synthetic procedure should not only be 28. J Wang, E Palecek, P E Nielsen, G Rivas, X Cai, H Shiraishi, efficient, but also fast and as cheap as possible. Examples of both N Dontha, D Luo, P A M Farias J. Am. Chem. Soc. 118 7667 (1996) the approaches to PNA synthesis have been presented in this 29. T J Griffin, L M Smith Anal. Biochem. 260 56 (1998) 30. U Giesen, W Kleider, C Berding, A Geiger, H Orum, P E Nielsen In low-budget laboratories, where the primary goal is eco- nomic PNA synthesis, it is the `slow' synthesis that is most 31. D Y Cherny, B P Belotserkovskii, M D Frank-Kamenetskii, M Egholm, O Buchardt, R H Berg, P E Nielsen Proc. Natl. Acad.
commonly used. Although this procedure is rather laborious, it gives excellent yields in the condensation step.
32. P E Nielsen, M Egholm, O Buchardt J. Mol. Recognit. 7 165 (1994) `Fast' processes are utilised in the majority of large biotechno- 33. P E Nielsen Methods Enzymol. 340 329 (2001) logical companies which manufacture PNA oligomers for com- 34. E Ferrer, A Shevchenko, R Eritja Bioorg. Med. Chem. 8 291 (2000) mercial purposes. Here, large excesses of PNA monomers and the 35. L Betts, J A Josey, J M Veal, SR Jordan Science 270 1838 (1995) most potent activating reagents are employed in order to ensure 36. R Gambari Curr. Pharm. Des. 7 1839 (2001) high yields of the condensation products. However, quantitative 37. P E Nielsen, M Egholm Bioorg. Med. Chem. 9 2429 (2001) yields cannot be attained due to a reduction of the condensation 38. Yu N Kosaganov, D A Stetsenko, E N Lubyako, N P Kvitko, time; therefore, pure PNA oligomers can be obtained by virtue of 39. M Egholm, L Christensen, K Dueholm, O Buchardt, J Coull, The most rational synthetic strategies combine the best P E Nielsen Nucl. Acids Res. 23 217 (1995) features of both approaches, viz., the `fast' and the `slow' syntheses 40. V V Demidov, M V Yavnolovich, B P Belotserkovskii, M D Frank-Kamenetskii, P E Nielsen Proc. Natl. Acad. Sci. USA 92 This work has been written within the framework of the State Programme for Support of Leading Scientific Schools of the 41. P Wittung, P E Nielsen, B Norden J. Am. Chem. Soc. 118 7049 Russian Federation (Grant No. 00-15-97944).
42. V V Demidov, M V Yavolovich, M D Frank-Kamenetskii 44. H Kuhn, V V Demidov, P E Nielsen, M D Frank-Kamenetskii 1. P E Nielsen, M Egholm, R H Berg, O Buchardt Science 254 1497 45. M C Griffith, L SRisen, M J Greig, E A Lesnik, K G Sprankle, 2. M Egholm, O Buchardt, L Christensen, C Behrens, SM Freier, R H Griffey, J SKiely, SM Freier J. Am. Chem. Soc. 117 831 (1995) D A Driver, R H Berg, SK Kim, B Norden, P E Nielsen Nature 46. SE Hamilton, M Iyer, J C Norton, D R Corey Bioorg. Med. Chem.
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14. B Hyrup, P E Nielsen Bioorg. Med. Chem. 4 5 (1996) 57. P E Nielsen, M Egholm, in Peptide Nucleic Acids: Protocols and 95. T Koch, H F Hansen, P Andersen, T Larsen, H G Batz, Applications. Synthesis of PNA Oligomers by Fmoc Chemistry K Ottesen, H Orum. J. Pept. Res. 49 80 (1997) (Eds P E Nielsen, M Egholm) (Wymondham: Horizon Scientific 96. D W Will, G Breipohl, D Langner, J Knolle, E Uhlmann 58. P E Nielsen Antiviral News 1 37 (1993) 97. SA Thomson, J A Josey, R Cadilla, M D Gaul, C F Hassman, 59. P E Nielsen, H Orum, in Molecular Biology: Current Innovations and M J Luzzio, A J Pipe, K L Reed, D J Ricca, R W Wiethe, Future Trends (Eds A M Griffin, H G Griffin) (Wymondham: 98. R B Merrifield J. Am. Chem. Soc. 85 2149 (1963) 60. M Egholm, P E Nielsen, O Buchardt, R H Berg, in Innovations and 99. K L Dueholm, M Egholm, C Behrens, L Christensen, Perspectives in Solid Phase Synthesis. Peptides, Proteins and Nucleic H F Hansen, T Vulpius, K H Petersen, R H Berg, P E Nielsen, Acids. Biological and Biomedical Applications (Ed. R Epton) (Birmingham: Mayflower Worldwide, 1994) p. 145 100. L Christensen, R Fitzpatrick, B Gildea, B Warren, J Coull, in 61. P E Nielsen, in Perspectives in Drug Discovery and Design Vol. 4 Innovations and Perspectives in Solid Phase Synthesis. Peptides, Proteins and Nucleic Acids. Biological and Biomedical Applications 62. A Ray, B Norden FASEB J. 14 1041 (2000) (Ed. R Epton) (Birmingham: Mayflower Worldwide, 1994) p. 149 63. P E Nielsen Methods Enzymol. 313 156 (2000) 101. L A Carpino Acc. Chem. Res. 20 401 (1987) 64. P E Nielsen Pharmacol. Toxicol. 86 3 (2000) 102. P Kocienski Protecting Groups (Stuttgart: Georg Thieme, 1994) 65. H Knudsen, P E Nielsen Anti-Cancer Drugs 8 113 (1997) 103. E Sonveaux, in Protocols for Oligonucleotides Conjugates, Methods 66. P E Nielsen, M Egholm, R H Berg, O Buchardt, in Antisense in Molecular Biology Vol. 26 (Ed. SAgrawal) (Totowa: Humana Research and Applications (Eds SCrook, B Lebleu) (Boca Raton, 104. Z Timar, L Kovacs, G Kovacs, Z Schmel J. Chem. Soc., Perkin.
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71. A De Mesmaeker, K-M Altman, A Waldner, SWendeborn Curr.
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80. A Beletskii, Y-K Hong, J Pehrson, M Egholm, W M Strauss Proc.
114. G Breipohl, J Knolle, D Langner, G Omalley, E Uhlmann Bioorg.
81. C Mischiati, M Borgatti, N Bianchi, C Rutigliano, M Tomassetti, 115. G Kovacs, Z Timar, Z Kele, L Kovacs, in The Fourth International G Feriotto, R Gambari J. Biol. Chem. 274 33 114 (1999) Electronic Conference on Synthetic Organic Chemistry (ECSOC-4), 82. H SMisra, P K Pandey, M J Modak, R Vinayak, V N Pandey 116. V A Efimov, M V Choob, A A Buryakova, A L Kalinkina, 83. J Weiler, H Gausepohl, N Hauser, O N Jensen, J D Hoheisel O G Chakhmakhcheva Nucl. Acids Res. 26 566 (1998) 117. M Eriksson, L Christensen, J Schmidt, G Haaima, L Orgel, 84. X Liu, SBalasubramanian Tetrahedron Lett. 41 6153 (2000) 85. SE Hamilton, C G Simmons, I SKathiriya, D R Corey Chem.
118. B Due Larsen, C Larsen, A Holm, in Peptides 1990, Proceedings of the 21st European Peptide Symposium (Eds E Giralt, D Andreu) 86. J Lohse, P E Nielsen, N Harrit, O Dahl Bioconj. Chem. 8 503 119. E Bayer, C Goldammer, in Peptides, Proceedings of the 12th 87. O Seitz, F Bergmann, D Heindl Angew. Chem., Int. Ed. Engl. 38 American Peptide Symposium (Eds J A Smith, J E Rivier) (Leiden: 88. F Lesignoli, A Germini, R Corradini, SSforza, G Galaverna, 120. SM Meister, SB H Kent, in Peptides Ð Structure and Function: A Dossena, R Marchelli J. Chrom. A 922 177 (2001) Proceedings of the Eighth American Peptide Symposium (Eds 89. T J Griffin, W Tang, L M Smith Nat. Biotechnol. 15 1368 (1997) V J Hruby, D H Rich) (Rockford, IL: Pierce Chem., 1984) p. 103 90. C Carlsson, M Jonsson, B Norden, M T Dulay, R N Zare, 121. C Mapelli, M D Sverdloff, in Peptides 1990, Proceedings of the 21st J Noolandi, P E Nielsen, L C Tsui, J Zielenski Nature (London) European Peptide Symposium (Eds E Giralt, D Andreu) (Leiden: 91. E Palecek, M Fojta, M Tomschik, J Wang J. Biosens. Bioelectron.
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Arrêté 2001-2376

Arrêté n° 276 CM du 29 mars 1994 fixant les conditions particulières de travail applicables aux femmes et aux jeunes travailleurs ainsi que les travaux présentant des causes de dangers ou excédant leurs forces et qui sont interdits aux jeunes travailleurs de moins de dix-huit ans et aux femmes Le Président du Gouvernement de la Polynésie française Sur le rapport du Minist


pTRE-Myc Vector Information GenBank Accession #: Submission in progress. EcoR I BamH I pTRE-Myc ATG GCA TCA ATG CAG AAG CTG ATC TCA GAG GAG GAC CTG CTT ATG GCC ATG GAG GCC CAA GCT TGG TCG ACC GAG ATC TCT CGA GGT ACC GCG GCC GCT CGA CGA TAT CTC TAG A Map and Multiple Cloning Site (MCS) of pTRE-Myc Vector. Unique restriction sites are in bold. Description: pTRE-Myc is a tetracyclin

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