The patent's assignee for patent number 8816062 is Universitat de
News editors obtained the following quote from the background information supplied by the inventors: "At present there is a great interest in identifying and developing oligonucleotides which are useful in therapy and diagnostics. The use of oligonucleotides in gene therapy aims the inactivation of the genes involved in the process of a disease. There are several strategies of treatments with oligonucleotides.
"Antisense therapy uses oligonucleotides with the sequence complementary to the target gene mRNA, which activates a gene silencing mechanism. It can also be used for altering the transcription of the defective gene by modifying, for example, its introns and exons editing pattern.
"iRNA small molecules are also used for activating a gene silencing mechanism similar to that of the antisense therapy.
"Another possibility is to use oligodeoxyribonucleotides as a decoy for the factors required in the activation of target genes transcription. Transcription factors are bound to the decoys instead of the promoter of the defective gene, which reduces the expression of the target genes. Moreover, single stranded DNA oligonucleotides have been used for directing the shift of one single base inside the sequence of a mutant gene.
"On the other hand, nucleic acid fragments with a suitable label (such as DNA probes) are used in diagnostic for the specific hybridization to a nucleic acid to be detected. The specific sequence of the new double strand is visualized with the aid of the label. Thus, genetic, carcinogenic, viral, or diseases caused by other pathogen agents can be detected.
"For the applications mentioned above, there are several limitations associated with the targeting to the specific cell, transport across the cell membrane and oligonucleotide stability. In this way, when the oligonucleotide is administered with a therapeutic or diagnostic purpose, sometimes the result obtained is much lower than expected, since it either does not reach the target cell, or it is not able to pass through the membrane, or it breaks down.
"In recent years protocols have been developed with the purpose of overcoming such limitations. These protocols are based on conjugating the oligonucleotide to a molecule which targets, specifically, the oligonucleotide to the target cell, which facilitates the transport across the cell membrane or which stabilizes the oligonucleotide. Examples of molecules that can be used with this purpose are, among others, cell penetrating peptides, lipids or polyamines (cf.
"Numerous protocols by means of which oligonucleotides are conjugated to agents of the type mentioned above are known in the state of the art. In most of these protocols, one of the steps consists of derivatizing the oligonucleotide with a functional group. This derivatization step is needed for being able to generate, in subsequent steps, the oligonucleotide-agent conjugate. In this derivatization step maleimide may be used as a functional group, thus obtaining maleimide-oligonucleotide derivatives. Maleimide derivatization allows the subsequent conjugation of any agent including a nucleophilic group, such as a thiol, or a diene.
"To date, the processes disclosed in the state of the art for preparing maleimide-oligonucleotide derivatives take place in solution (cf. Harrison G. H. et al., 'Synthesis and hybridization analysis of a small library of peptide-oligonucleotide conjugates', Nucleic Acids Res. 1998, vol. 26, pp. 3136-3145; Zanta M. A. et al., 'Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus', Proc. Natl. Acad. Sci. 1999, vol. 96, pp. 91-96). The processes described for obtaining maleimide-oligonucleotide derivatives show regioselectivity problems due to the fact that they are performed in solution. This negatively affects the purity of the resulting maleimide-oligonucleotide derivative and, in turn, the yield of such processes, since the amount of the derivative finally obtained is reduced as being necessary subsequent steps for its purification. The fact that these processes result in derivatives with low yield and purity affects the subsequent steps, wherein the oligonucleotide, functionalized with the maleimide, is conjugated to the agent of interest (peptide, protein, etc.) in such a way that the oligonucleotide has the desired therapeutic or diagnostic effect. Starting from a small amount of the maleimide-oligonucleotide derivative, a much lower final amount of the oligonucleotide with therapeutic or diagnostic activity to that initially expected is obtained.
"Therefore, there is a need for providing processes which allow to obtain maleimide-oligonucleotide derivatives with suitable yield and purity."
As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors' summary information for this patent: "The inventors of the present invention have developed a general process for preparing maleimide-oligonucleotide derivatives in solid-phase which is regioselective. This process comprises, in a first step, coupling a maleimide group to an oligonucleotide of interest (having an intended nanotechnologic, diagnostic or therapeutic application), which is immobilized on a solid support. The inventors have confirmed that when the maleimide group is protected by a furanyl portion, thus forming a new furanyl-maleimide compound, the coupling step is regioselective. Consequently, the general process is automatable, proceeds with high yields and allows to obtain, at the end of the process, a high purity maleimide-oligonucleotide derivative.
"A second step of the general process for preparing maleimide-oligonucleotide derivatives of the invention is the release of the oligonucleotide derivative from the support. In this regard, the inventors have found that the furanyl-maleimide compound has to be substantially in the exo configuration since, unlike the endo isomer, the exo isomer is stable under the conditions in which the oligonucleotide is released from the support.
"Additionally, the inventors of the present invention have found that using the furanyl-maleimide compound of the invention, which is characterized by comprising methyl groups in positions 2 and 5 of the furanyl portion, the retro-Diels-Alder type reaction may be carried out under milder conditions. This reaction is the last step of the general process of the invention and is carried out in order to finally obtain the maleimide-oligonucleotide derivative of interest.
"Due to the foregoing, the furanyl-maleimide compound designed by the inventors of the present invention is a key intermediate in the general process for the invention.
"Thus, a first aspect of the present invention relates to a compound of formula (I) substantially in exo configuration, or a salt thereof:
"##STR00002## wherein: X is a biradical selected from the group consisting of: --(CH.sub.2).sub.n--*,--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--*,
"##STR00003## n is an integer ranging between 1 and 30; * representing the place through which X binds to Y; Y is a radical selected from the group consisting of:
"##STR00004## the wavy line representing the place through which Y binds to X; PG is a phosphate protecting group; and R.sub.1 and R.sub.2 are the same or different to each other and are selected from a C.sub.1-C.sub.10 alkyl radical and a morpholine radical.
"As it has been stated above, the general process for preparing the maleimide-oligonucleotide derivatives of the invention is regioselective. This regioselectivity is due, on one hand, to the fact that the maleimide portion is protected by the furanyl portion and, on the other hand, to the fact that the derivatization process is carried out in solid phase.
"The compound of formula (I) according to the first aspect of the invention is characterized by having the maleimide portion protected with a furanyl portion. In this way, when the compound of formula (I) reacts with the oligonucleotide (with the purpose of derivatizing it), the reaction takes place through a single point in the maleimide portion (i.e., through the Y radical of the compound of formula (I)), thus avoiding secondary reactions due to the existence of reactivity in other positions of the maleimide portion.
"Furthermore, the fact that the oligonucleotide is immobilized on the support assists the derivatization to take place through the desired point of the oligonucleotide. Regioselectivity of the derivatization step may be improved if the oligonucleotide, which is immobilized on the solid support, has the reactive groups (i.e., exocyclic amino, phosphates, and OH groups of the bases) blocked, except the reactive group of the oligonucleotide through which the derivatization reaction with the compound of formula (I) is intended to take place. Protecting groups well-known in the state of the art can be used for blocking the exocyclic amino groups, phosphate groups and hydroxyl groups.
"In a second aspect, the present invention relates to a process for preparing the compound of formula (I) defined in the first aspect of the invention, wherein Y is --COOH, comprising the steps of: (a) carrying out a Diels-Alder reaction between a compound of formula (II) and a compound of formula (III),
"##STR00005## wherein X is as defined above, and (b) carrying out a treatment of the compound obtained in step (a) with a nucleophilic base for isolating the compound of formula (I).
"In a third aspect, the present invention relates to a process for preparing the compound of formula (I) defined in the first aspect of the invention, wherein Y is
"##STR00006## wherein the wavy line, PG, R.sub.1 and R.sub.2 are as defined above, comprising the reaction of a compound of formula (IV) with a compound of formula (V):
"##STR00007## in an aprotic solvent and under anhydrous conditions, wherein Z is selected from halogen and diisopropylamine, and X is as defined above.
"In a fourth aspect, the present invention relates to a process for solid-phase preparation of a maleimide-oligonucleotide derivative of formula (VI),
"##STR00008## wherein X is as defined in the first aspect of the invention, and Y' is selected from
"##STR00009## wherein the wavy line represents the place through which Y' binds to X, # represents the place through which Y' binds to the oligonucleotide and PG is as defined above; the process comprising the following steps: (a) coupling the compound of formula (I) of the first aspect of the invention to an oligonucleotide that is immobilized on a solid support, in order to obtain the compound of formula (VII)
"##STR00010## wherein P is the solid support, (b) releasing the compound of formula (VII), resulting from step (a), from the solid support to give rise to the compound of formula (VIII);
"##STR00011## and © subjecting the compound of formula (VIII) to a retro-Diels-Alder reaction, such that the derivative of formula (VI) is obtained.
"As stated above, the compound of formula (I) and the fact that the coupling to the oligonucleotide takes place in a solid phase confer regioselectivity to the preparation process of the fourth aspect of the invention.
"The compound of formula (I) of the invention, depending on the spatial arrangement of its atoms, may adopt an exo or endo configuration. According to the present invention, the compound of formula (I) is found substantially in an exo configuration, which means that it has a percentage of exo isomer equal to or greater than 95%, more preferably equal to or greater than 98%.
"The inventors have found that the compound of general formula (I) is stable under the conditions necessary for obtaining the compound (VIII) from the compound (VII). This stability is conferred by the fact that the compound of the invention is substantially in exo configuration. As it is illustrated below, when using ammonia, which is a frequently used reactive for releasing an oligonucleotide from a solid support, it is observed that if a mixture of exo/endo product is used, the recovered product is the one corresponding to the exo adduct, while the endo form breaks down resulting in undesired secondary products. The experimental results obtained allow to conclude that using a compound of formula (I), which is found substantially in exo configuration, a purer compound (VIII) is obtained, since the by-products associated with the degradation of the endo isomer are minimized, and the final yield of the maleimide-oligonucleotide derivative is also higher.
"On the other hand, the inventors have confirmed that the fact that the furanyl portion is substituted in positions 2 and 5 with methyl groups allows to carry out the retro-Diels-Alder-type reaction of step © under milder conditions. Being able to work under milder conditions minimizes the risk of degradation of the oligonucleotide, which, in turn, contributes to a higher yield of the process.
"As it has already been explained above, the production of maleimide-oligonucleotide derivatives is an intermediate step necessary for the oligonucleotide to end up conjugating to the agent which confers cell specificity, stability, or the ability of being transported across the cell membrane (cf. Lonnberg H. et al., supra).
"The fact that the maleimide-oligonucleotide derivative of formula (VI) of the present invention is obtained in a regioselective manner and with suitable yield and purity positively affects the subsequent generation of the oligonucleotide-agent conjugates (which are the ones having the intended nanotechnologic, therapeutic or diagnostic application). The purer the maleimide-oligonucleotide derivative, less by-products will be obtained during the conjugation of the agent to the derivative, which positively affects the final yield of the oligonucleotide-agent product since additional purification steps will not be needed.
"In a fifth aspect, the present invention relates to a compound of formula (VII)
"##STR00012## wherein X, Y' and P are as defined above.
"In a sixth aspect, the present invention relates to a compound of formula (VIII):
"##STR00013## wherein X and Y' are as defined above."
For additional information on this patent, see: Grandas Sagarra, Ana Maria;
Keywords for this news article include: Carboxylic Acids, Cell Membrane, Cellular Structures, Maleates, Maleimides, Organic Chemicals, Peptides, Proteins, Proteomics, Therapy, Universitat de
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