Easy introduction of maleimides at different positions of oligonucleotide chains for conjugation purposes

[2,5-dimethylfuran]-protected maleimides were placed at both internal positions and the 3'-end of oligonucleotides making use of solid-phase synthesis procedures. A new phosphoramidite derivative and a new solid support incorporating the protected maleimide moiety were prepared for this purpose. In all cases maleimide deprotection (retro-Diels–Alder reaction) followed by reaction with thiol-containing compounds afforded the target conjugate. 10


Introduction
Derivatization of oligonucleotides by covalent attachment of a non-oligonucleotide moiety, usually referred to as conjugation, is a common alternative to improve oligonucleotides performance in biological assays and suitability for therapeutic applications (1).3'-Modification is known to impart protection to the most ubiquitous nucleases, namely 3'-exonucleases (2), and conjugation often improves oligonucleotides' cell permeation properties.In general there is a rationale on the decision as to which moiety is to be linked to the oligonucleotide chain, but the linking site and the linkage type are most often dictated by the chemically available tools (resins or phosphoramidite derivatives commercially available, derivatives synthesized at the laboratory carrying out the study, etc.).Even though changes in the constitution of the conjugate will likely have an influence on the outcome of biological assays, there is not much information available on this type of structure-activity correlation.Indeed, there are reports showing the outcome of such changes.It has been described, for instance, that the immune stimulating properties of conjugates in which a 28-mer !-amyloid peptide was attached to an oligonucleotide varied depending on whether the peptide was linked to either the 5'-or the 3'-end (3).It has also been reported that the stability of thiol-maleimide adducts may vary depending on the "external" environment, such as the presence of inorganic anions (4) or reducing agents (thiols) (5), but also depending on the structure of the conjugate itself (6).In a recent piece of work (6), Junutula and co-workers have shown that the stability and activity of antibody-drug conjugates varies depending on whether the conjugation site is placed in a highly solvent accessible and positively charged local environment, or in a partially accessible and neutral local environment.In a different context, but also showing the importance of conjugates' constitution, an interesting example is the use of DNA as a ruler (7), which shows that the covalent attachment of peptides to different positions of the chain can provide information on the spatial distribution of protein binding sites (7, see references therein for other examples of applications of complex molecular structures).Oligonucleotide conjugates can be assembled making use of 50 solid-phase technologies when suitably derivatized building blocks of solid supports are available (8).Convergent solution synthesis is the other alternative.In this case the two moieties to be linked must be derivatized with functional groups ideally reacting regio-and chemoselectively, exploiting the so-called 55 click chemistry (9).The par excellence click reaction is the Cu(I)catalyzed azide-alkyne cycloaddition, but well-performing click reactions can exploit other functional groups as well.Maleimides are examples of useful functional units, since they can be involved in two different click reactions, the Michael-type 60 reaction with thiols and the Diels-Alder cycloaddition.For many years the most common alternative for the derivatization of oligonucleotides with a maleimide has been the reaction, in solution, of bifunctional compounds incorporating a carboxylic acid and a maleimide with the amine group of amino-65 derivatized oligonucleotides (10).The problem with this methodology is that amide formation does not always take place in high yield.Yet, attachment of the maleimide-containing bifunctional compound to amino-derivatized resin-linked oligonucleotides is not an alternative, because the ammonia 70 treatment that removes oligonucleotide protecting groups degrades the maleimide (11).Solid-phase assembly is only possible provided that the maleimide is protected and remains stable to the ammonia deprotection treatment.We have recently described that 75 maleimide building blocks fulfilling this requirement can be obtained by reaction with 2,5-dimethylfuran, followed by removal of the ammonia-labile endo adduct (12).Bifunctional compounds containing a protected maleimide (exo adduct) and either phosphoramidite (1) or carboxyl (2) groups (Figure 1) can using the same chemistry as for all nucleosides.On the other, it is not necessary to assemble amine-derivatized resin-linked oligonucleotides to which 2 must be coupled by forming an amide bond.Even though this type of reactions takes place on a solid matrix, which allows large excesses of the activated carboxyl-containing species to be added, coupling yields have been described as poorly reproducible and not always fully satisfactory (13).Incorporation of a protected maleimido unit after oligonucleotide elongation using standard nucleoside-3'-phosphoramidite derivatives, followed by deprotection with ammonia and heating to provide the free maleimide (retro-Diels-Alder reaction), affords 5'-maleimido-oligonucleotides.Chain elongation with nucleoside-5'-phosphoramidites might allow 3'-maleimidooligonucleotides to be assembled, but these derivatives are much more expensive than standard nucleoside-3'-phosphoramidite.Since, as described above, it is important to have access to conjugates with different structures, we decided to prepare a new phosphoramidite building block allowing the maleimide unit to be placed at internal positions of the oligonucleotide sequence (in fact at any position of the chain), and a new solid matrix incorporating the protected maleimide moiety that is suitable for the synthesis of 3'-maleimido-oligonucleotides making use of 3'phosphoramidite derivatives.In this manuscript we describe their synthesis and their use in the preparation of maleimidooligonucleotides.These maleimido-oligonucleotides were reacted with thiols to assess the functionality of the maleimides.

Results and discussion
The synthesis of building block 5 and solid support 7 is summarized in Scheme 1.Both incorporate a DMT-protected hydroxyl to allow for further elongation of the chain.The two compounds 5 and 7 derive from common precursor 4, which has the advantage that preparation of 4 provides a stock of a product from which either 5 or 7 can be obtained in one or two steps.
For the synthesis of 4, the amino group of L-serinol, more nucleophilic than the hydroxyl groups, was derivatized first.Reaction between 2 (exo adduct), a carbodiimide and pentafluorophenol provided the pentafluorophenyl ester of 2, and this active ester reacted with L-serinol to form the amide with no need to protect the hydroxyl groups.Then, one of the hydroxyl groups of the resulting compound (3) was protected by reaction with DMT-Cl.Use of a smaller amount of DMT-Cl (0.9 equiv) with respect to 3 facilitated formation and isolation of 4 as the main product.Phosphitylation of 4 afforded phosphoramidite 5, while reaction of 4 with succinic anhydride and incorporation of the resulting 6 onto LCAA-CPG provided support 7. Derivatives 5 and 7 were used in the synthesis of two maleimidooligonucleotides, 9 and 12 (Scheme 2).In both cases chain elongation proceeded smoothly, showing that the two derivatives 55 behaved as standard building blocks.The [protected maleimido]oligonucleotides (8 and 11) obtained after reaction with ammonia at room temperature were purified by HPLC (Figure 2) and characterized by mass spectrometry.Maleimide deprotection was carried out using the method that 60 had performed better in our previous work (12,14), namely heating a suspension of the oligonucleotide in anh.toluene at 90 o C for 4 h.Maleimide deprotection yields were above 90 %, as assessed by HPLC analysis of the crudes (Figure 3).Finally, it was verified that reversal of the Diels-Alder dimethylfuran-maleimide cycloadduct afforded oligonucleotides with appending fully reactive maleimides.For this purpose, maleimido-oligonucleotides 9 and 12 were reacted with thiolcontaining compounds, as shown in Scheme 2. Michael-type 15 thiol-maleimide reactions yielded the target conjugates, as confirmed by mass spectrometric analysis of the product corresponding to the main peak of the HPLC trace of the crude (Figure 4).The maleimide moiety linked to the 3'-end of the oligonucleotide thoroughly reacted with two primary thiols, those 20 of biotin-SH and glutathione, to yield conjugates 13 and 14, respectively (Figure 4b,c).Likewise, reaction between the maleimide placed at an internal position and the secondary thiol of thiocholesterol, somehow more demanding because the reaction groups were more hindered, cleanly furnished conjugate 25 10 (Figure 4a).

Conclusions
In summary, a phosphoramidite derivative and a solid support 30 incorporating 2,5-dimethylfuran-protected maleimide moieties were synthesized from a common precursor (4), obtained from Lserinol.Both were satisfactorily used in the preparation of maleimido-oligonucleotides that subsequently underwent addition of thiols, thus proving that maleimide deprotection affords 35 reactive maleimides.This new building block and resin expand the repertoire of possibilities allowing the derivatization of oligonucleotides at different positions, and thus give access to differently decorated oligonucleotide chains.
TLC was carried out on silica gel plates 60 F 254 from Merck.Samples were lyophilized in a FreezeMobile Virtis instrument.
The amount of free thiols in thiol-containing compounds was quantified by the Ellman test, as described in the Supporting Information of reference 12.
Oligonucleotide synthesis.Oligonucleotide chains were assembled in a 3400 ABI automatic synthesizer at the 1 µmol scale, using standard phosphoramidite synthesis cycles.Phosphoramidite 5 and solid support 7 were used as any standard reagent.After chain elongation, treatment with concd.aq.toluene was added (the amount that would be required to obtain a 25 µM solution).The mixture was heated 90 ºC, toluene was removed under reduced pressure, and the crude was dissolved in water for HPLC analysis and mass spectrometric characterization.The crude was typically used at the subsequent conjugation step without any purification.General procedure for Michael-type conjugation reactions.Aliquots of aqueous solutions containing the required amounts of maleimido-containing oligomer and the corresponding thiolcontaining compound (5 to 10-fold molar excess) were mixed, 40 and the mixture was diluted with 0.5 M triethylammonium acetate, pH = 7.8-7.9(final concentration of oligomer: 50-150 µM).The mixture was stirred at room temperature under an Ar atmosphere.Reaction crudes were analyzed by HPLC.Conjugates were purified by HPLC and characterized by 45 MALDI-TOF MS.Synthesis of conjugate 10: thiocholesterol (not soluble in water) was dissolved in THF, and the reaction was carried out in a 3:2 (v/v) 0.5 M triethylammonium acetate (pH = 7.8)/THF mixture.50 HPLC.Reversed-phase HPLC analysis and purification was performed using analytical and semipreparative Waters or Shimadzu systems.Analysis and purification conditions were: Oligonucleotide analysis conditions: Kromasil C18 column (10 µm, 100 Å, 250 " 4.0 mm) from Akzo Nobel; solvent A: 0.05 M 55 triethylammonium acetate, solvent B: H 2 O/ACN 1:1 (v/v), gradient from 5 to 60 % of B in 30 min, flow: 1 mL/min, detection wavelength: 254 nm.Oligonucleotide purification conditions (semipreparative scale): Jupiter C18 column (10 µm, 300 Å, 250 " 10.0 mm) from 60 Phenomenex, solvent A: 0.1 M triethylammonium acetate, solvent B: H 2 O/ACN 1:1 (v/v), gradient from 5 to 60 % of B in 30 min, flow: 3 mL/min, detection wavelength: 260 nm.Cholesterol-containing conjugate analysis and purification conditions: Kromasil C4 column (10 µm, 100 Å, 250 " 4.6 mm) 65 from Teknokroma, solvent A: 0.05 M triethylammonium acetate (pH = 7.0), solvent B: acetonitrile, flow: 1 mL/min, detection wavelength: 254 nm.After injection of the samples, the column was eluted for 5 min with 10% of B, followed by gradient from 10 to 90 % of B in 25 min.Subsequently, the column was eluted 70 for 10 min with 90% of B. Mass spectrometry.MALDI-TOF mass spectra were recorded on a 4800 Plus ABSciex instrument using reflector.Oligonucleotide and conjugates analysis conditions: 1:1 (v/v) 2,4,6-trihydroxyacetophenone/ammonium citrate (THAP/CA), 75 negative mode.ESI (low and high resolution) mass spectra were obtained using an LC/MSD-TOF spectrometer from Agilent Technologies.

Synthesis of the [protected maleimido]-containing monomer 5
and solid support 7.

2 Figure 1 .
Figure 1.Structures of the [protected maleimido]-containing derivatives previously used in the synthesis of maleimido-oligonucleotides.