Carbamoylation of Amines, Thiophenols, Mercaptanes and Phenols Employing Organic Azides

Field of the Invention

The present invention relates to carbamoylation of amines, mercaptanes, thiophenols and phenols employing organic azides. More specifically, the invention relates to a method for generating urea derivatives, thiocarbamate derivatives and carbamate derivatives, and is based on the intermediate formation of isocyanate, starting from an organic azide. The reaction as described is useful in applications for modified nucleoside synthesis, oligonucleotide synthesis, as well as modification, labeling and conjugation of polymers and biomolecules.

Background of the Invention

International patent application No. WO 2005/061445 (Langstrom et al.) (1) and references cited therein are describing carbonylation via isocyanate using azides and carbon monoxide. This reaction is promoted by a transition metal complex (e.g rhodium, palladium) and is performed in a high pressure reaction chamber. The main features of Langstrom' s and similar methods are as follows: Introduction of carbon monoxide into the reaction chamber via the gas inlet and introduction at high pressure an azide solution mixed with a transition metal complex and a liquid reagent (solvent) into the reaction chamber via the liquid inlet. Since Langstrom method is dealing with carbon-isotope monoxide, additional technical measures have to be undertaken for trapping the carbon- isotope dioxide and converting it to carbon-isotope monoxide.

Obviously, these reactions require very special equipment, alkyl azide solution, expensive transition metal complex and hazardous highly toxic gas - carbon monoxide.

In contrast to this kind of procedure, the present method utilizes an alkyl azide solution, inexpensive compound of trivalent phosphorous (e.g. triphenylphosphine) and trialkyl- ammonium hydrogen carbonate buffer. This buffer is prepared by simple bubbling of harmless carbon dioxide in a mixture of trialkylamine and water until pH about 7-8 is

reached. The carbamoylation reaction itself is then performed in a tightly closed vessel, like, e.g, a bottle with a screw cap.

It is noteworthy that the present procedure is extremely simple. It does not require any special equipment (unlike Langstrom's (1) or similar procedures), any expensive transition metal complexes or, more importantly, a hazardous highly toxic gas - carbon monoxide. In other words, the present procedure may be carried out in any chemical laboratory.

Summary of the Invention

The present invention relates to a straightforward method of carbamoylation of amines, mercaptanes, thiophenols and phenols, employing an organic azide, a compound of trivalent phosphorous, an aqueous trialkylammonium hydrogen carbonate buffer and an organic solvent. This method may be successfully employed in basic organic chemistry, and also for the synthesis of various nucleoside derivatives and modification of various particles and solid surfaces.

Brief Description of Drawings

Figure 1 shows transformations of 3'-azido-3'-deoxythymidine.

Figure 2 shows modification of 2'-amino-2'-deoxynucleosides.

Figure 3 shows modification of polystyrene and/or controlled pore glass based solid supports resulting in Universal Solid Supports for oligonucleotide synthesis (6,7).

Figure 4 shows modification of polystyrene and/or controlled pore glass based solid supports resulting in Nucleoside-bound solid supports for oligonucleotide synthesis (8).

Figure 5 schematically shows azidoalkyl-tethered synthetic oligonucleotide for subsequent attachment to solid phases and surfaces and fluorescein- labeled synthetic oligonucleotides for testing oligonucleotide-derivatized nanoparticles and microarray slides.

Figure 6 shows oligonucleotide-functionalized nanoparticles.

Figure 7 shows an oligonucleotide- functionalized microarray slide.

Detailed Description of the Invention

Abbreviations

Ade Adenin-9-yl

AMPS Macroporous Amino methyl Polystyrene

CPG Controlled Pore Glass

Cyt Cytosin-1-yl

DMTr 4,4'-Dimethoxytriphenylmethyl

Gua Guanin-9-yl

Fmoc 9-Fluorenylmethoxycarbonyl

N ⁶ -Bz-Ade //"-Benzoyl- Adenin-9-yl

N ⁴ -Bz-Cyt //-Benzoyl-Cytosin- 1 -yl

N ² -ibu-Gua jV ² -isobutyryl-Guanin-9-yl

Thy Thymin-1-yl

Ura Uracil- 1-yl

USUI Universal Solid Support III

The present invention relates to the reaction of carbamoylation of amines Ia, mercaptanes Ib, thiophenols Ic or phenols Id, employing organic azides II (Scheme 1). The reaction proceeds via intermediate formation of isocyanates of general formulae III and results in products of general formulae IV.

Basic chemistry of various transformations mentioned herein is depicted as follows from Scheme 2.

The organic group R in Schemes 1 and 2 may be any organic group capable of forming an organic azide compound. Consequently, R may be linear or cyclic lower alkyl, which may optionally be substituted, arylalkyl, aminoalkyl, or lower alcohol. R may also be nucleosidyl, nucleotidyl, oligonucleotidyl or peptidyl, as well as ribosyl, 2'-deoxyribosyl or

any functional derivative thereof. In any of the mentioned organic groups any functional group may be protected, if appropriate. Preferably R is lower aminoalkyl or nucleosidyl, more preferably 3-aminopropyl or 3'-deoxythymidilyl.

R' as an aliphatic organic group is preferably linear or cyclic lower alkyl, which is optionally substituted, or deoxynucleosidyl. R is in this case, for instance, hydroxyethyl.

R as an aromatic organic group is preferably aryl or substituted aryl. R is in this case, for instance, phenyl or benzyl.

The method of synthesis described in the present application comprises reduction of organic azides II with a compound of trivalent phosphorous (triphenylphosphine, trialkyl- phosphine, trialkylphosphite, hexaalkyltriamidophosphite, etc.) in an organic solvent (1,4- dioxane, tetrahydrofurane, acetonitrile, etc.) in the presence of hydrogen carbonate ions (various trialkylammonium hydrogen carbonate buffers, e.g. trimethylammonium hydrogen carbonate, triethylammonium hydrogen carbonate, diethyl-2-hydroxyethyl- ammonium hydrogen carbonate, etc.), leading to formation of intermediate structures incorporating -P=N- function, followed by formation of isocyanates III and finally by reaction with amines, mercaptanes, thiophenols or phenols as nucleophiles to give rise to ureas IVa, thiocarbamates IVb and IVc or carbamates IVd.

The procedure to generate substituted ureas IVa, thiocarbamates IVb,c and carbamates IVd is the preferred method of the present invention by virtue of its broad employment for synthesis and modification of various organic compounds.

Since the intermediate reactive product of this reaction is an isocyanate of structure III, the present invention may be successfully utilized in chemical synthesis and chemical industry, where generation of isocyanates is required or where isocyanates serve as starting compounds. The present invention discloses a procedure which complements a number of contemporary methods of synthesis and manufacture of isocyanates (2, 3, 4, 5).

The procedure is a highly effective and simple new conjugation reaction that is complementing conventional methods of bioconjugation. It is applicable in diverse areas

including applications for oligonucleotide synthesis, modification and conjugation. More broadly it may find use in nano techno logy, arrays, diagnostics and screening assays. The technique can be readily engineered to link small molecules (peptides, fluorophores, oligonucleotides, etc.), biomolecules (proteins, DNA, RNA, antibodies), or other molecules to solid surfaces (beads, glass, plastic, latex), for applications in proteomics, genomics, drug discovery, diagnostics and therapeutics. The present invention will also enable the development of new applications in both genomics and proteomics that cannot be satisfied with current conventional methods.

Advantages of the present technology include: simple and easy-to-use protocol; carrying out the reactions at room temperature in an organic solvent containing aqueous buffered media and yielding a high-efficiency conjugation; obtaining conjugates which have extended stability.

Consequently, the present invention may be utilized in processes in which generation of isocyanates is required or where isocyanates serve as starting compounds to react with aminoalkyl, mercaptoalkyl, thiophenylalkyl and hydroxyphenylalkyl functions.

In more detail, the present invention allows to generate the above-mentioned structures as bridges for:

conjugation of molecules bearing azidoalkyl tethers with molecules bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl or hydroxyphenylalkyl groups; conjugation of molecules bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl or hydroxyphenylalkyl tethers with molecules bearing azidoalkyl groups; conjugation of nucleosides, nucleotides and oligonucleotides bearing azidoalkyl tethers with various molecules bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl or hydroxyphenylalkyl groups (luminescent and spin labels, various chelates, modified peptides, modified proteins, modified antibodies, etc.); conjugation of nucleosides, nucleotides and oligonucleotides bearing azidoalkyl tethers with peptides, proteins, antibodies, etc.;

conjugation of molecules (luminescent and spin labels, various chelates, etc.) bearing azidoalkyl tethers with peptides, proteins, antibodies, etc.; conjugation of nucleosides, nucleotides and oligonucleotides bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl or hydroxyphenylalkyl tethers with various molecules bearing azidoalkyl groups (luminescent and spin labels, various chelates, modified peptides, modified proteins, modified antibodies, etc.); conjugation of oligonucleotides bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl or hydroxyphenylalkyl tethers with solid phase bearing azidoalkyl groups to prepare oligonucleotide arrays, oligonucleotide-bound microparticles, nanoparticles, etc.; conjugation of oligonucleotides bearing azidoalkyl tethers with solid phase bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl and hydroxyphenylalkyl groups to prepare oligonucleotide arrays, oligonucleotide-bound microparticles, nanoparticles, etc.; conjugation of protected nucleosides bearing azidoalkyl tethers with various solid matrices (controlled pore glass, polystyrene, polyvinylacetate) bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl or hydroxyphenylalkyl groups to prepare nucleoside-bound solid supports for DNA, RNA and modified oligonucleotide solid phase synthesis; conjugation of protected nucleosides bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl and hydroxyphenylalkyl tethers with various solid matrices (controlled pore glass, polystyrene, polyvinylacetate) bearing azidoalkyl groups to prepare nucleoside-bound solid supports for DNA, RNA and modified oligonucleotide solid phase synthesis; conjugation of specific molecules bearing azidoalkyl tethers with various solid matrices (controlled pore glass, polystyrene, polyvinylacetate) bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl and hydroxyphenylalkyl groups to prepare universal solid supports for DNA, RNA and modified oligonucleotide solid phase synthesis; conjugation of specific molecules bearing aminoalkyl, mercaptoalkyl, thiophenylalkyl and hydroxyphenylalkyl tethers with various solid matrices (controlled pore glass, polystyrene, polyvinylacetate), bearing azidoalkyl groups to prepare universal solid supports for DNA, RNA and modified oligonucleotide solid phase synthesis.

Examples

1. 3 '-Azido-3 '-deoxy thymidine derivatives

Example 1

3 '-Azido-3 '-deoxythymidine (1, 0.37 mmol) was added to a solution of triphenyl- phosphine (0.4 mmol) in a mixture of dioxane (4 ml) and IM aqueous triethyl- ammonium hydrogen carbonate (0.5 ml). The mixture was left for 24 hours at room temperature and evaporated to dryness. Chromatographic separation on silica gel afforded dimer 2 (Figure 1) in 53% yield.

Example 2

3 '-Azido-3 '-deoxythymidine (1, 0.37 mmol) was added to a solution of 1 mmol of compound benzylamine (3) or thiophenol (4) or mercaptoethanol (5) or phenol (6) and triphenylphosphine (0.4 mmol) in a mixture of dioxane (4 ml) and IM aqueous triethylammonium hydrogen carbonate (0.5 ml). The mixture was left for 4 hours (for compounds 7-9) or for 24 hours (for compound 10) at room temperature and evaporated to dryness. Chromatographic separation on silica gel afforded compounds 7-9 in about 90% yield; compound 10 in 5% yield (Figure 1).

Example 3

3 '-Azido-3 '-deoxythymidine (1, 0.37 mmol) was added to a solution of 1 mmol of compound benzylamine (3) and 1 mmol of mercaptoethanol (5) and triphenylphosphine (0.4 mmol) in a mixture of dioxane (4 ml) and IM aqueous triethylammonium hydrogen carbonate (0.5 ml). The mixture was left for 12 hours at room temperature and analyzed with RP HPLC. The HPLC trace and integration of peaks revealed the complete conversion of azide 1 to give compounds 7 and 9 in 2:1 ratio (Figure 1).

2. Modification of aminonucleosides

Example 4

2'-Amino-2'-deoxynucleoside (lla-d, 0.37 mmol) was added to a solution of 1 mmol of azide 12 and triphenylphosphine (0.4 mmol) in a mixture of dioxane (4 ml) and IM aqueous triethylammonium hydrogen carbonate (0.5 ml). The mixture was left for 24 hours at room temperature and evaporated to dryness. Chromatographic separation on silica gel afforded compounds 13a-d in about 80% yield (Figure 2).

3. Modification of particles and solid surfaces:

A. Modification of polystyrene and controlled pore glass based solid supports resulting in Universal solid supports for oligonucleotide synthesis (6,1).

Example 5

A solution of azide 14 in dioxane (11.5 ml of 0.09 M solution for 0.4 mmol of linker loaded support; 23 ml of 0.09 M solution for 0.8 mmol of linker loaded support) was added to a suspension of 20 g of Macroporous Aminomethyl polystyrene (cross-linking - 60%, particle size - 100-200 mesh, loading of amino groups - 0.12 mmol/g) in dioxane (188 ml for 0.4 mmol of linker loaded support; 177 ml for 0.8 mmol of linker loaded support). To the resulting suspension the aqueous solution of triethylammonium hydrogen carbonate (2 M, 5 ml) and triphenylphosphine (3 g for 0.4 mmol of linker loaded support; 6 g for 0.8 mmol of linker loaded support) were added and the mixture was shaken for 48 h at room temperature. The resin was filtered off, washed with acetone, followed by tetrahydrofurane and re-suspended in tetrahydrofurane (50 ml). A mixture of pyridine (70 ml) and acetic anhydride (30 ml) was then added and the resulting suspension was left for 3 h at room temperature with periodic shaking. The resin was filtered off, washed with pyridine (30 ml), acetone (200 ml), 0.1% triethylamine in ether and finally dried in high vacuum. The resulting dry resin, contained either about 0.04 mmol of DMTr-groups per gram of polymer (8) - (USIII- AMPS-40) (Figure 3), or about 0.08 mmol of DMTr-groups per gram of polymer (8) - (USIII-AMPS-80) (Figure 3). Both polymers, Universal Solid Supports for Oligonucleotide synthesis (USIII-AMPS-40 and USIII-AMPS-80), performed identically to the Universal Solid Support, described in detail earlier (6,7).

Example 6

A solution of azide 14 in dioxane (11.5 ml of 0.09 M solution) was added to a suspension of 20 g of Aminoalkyl Controlled Pore Glass (CPG-500: particle size - 120-200 mesh, loading of amino groups - 0.12 mmol/g, pore diameter 5OθA or CPG- 1000: particle size - 120-200 mesh, loading of amino groups - 0.06-0.07 mmol/g, pore diameter lOOOA) in dioxane (188 ml). To the resulting suspension the aqueous solution of triethylammonium hydrogen carbonate (2 M, 5 ml) and triphenylphosphine (3 g) were added and the mixture was shaken for 48 h at room temperature. The resin was filtered off, washed with acetone, followed by tetrahydrofurane and re-suspended in tetrahydrofurane (50 ml). A mixture of pyridine (70 ml) and acetic anhydride (30 ml) was then added and the resulting suspension was left for 3 h at room temperature with periodic shaking. The solid phase was filtered off, washed with pyridine (30 ml), acetone (200 ml), 0.1% triethylamine in ether and finally dried in high vacuum. The resulting dry solid phase contained: about 0.04 mmol of DMTr-groups per gram of CPG-500 (8) - USIII-CPG-500-40 (Figure 3), or about 0.03 mmol of DMTr-groups per gram of CPG-1000 (8) - USIII-CPG- 1000-30 (Figure 3). Both solid phases, Universal Solid Supports for Oligonucleotide synthesis (USIII-CPG-500-40 and USIII-CPG- 1000-30), performed identically to the Universal Solid Support, described in detail earlier (6,7).

Example 7

Aminoalkyl Controlled Pore Glass (CPG-500: particle size - 120-200 mesh, loading of amino groups - 0.12 mmol/g, pore diameter 5OθA or CPG-1000: particle size - 120— 200 mesh, loading of amino groups - 0.06-0.07 mmol/g, pore diameter lOOOA) or Macroporous Aminomethyl polystyrene (cross-linking - 60%, particle size - 100-200 mesh, loading of amino groups - 0.12 mmol/g) were derivatized with 3'-O-(4- azidobutyryl)-5'-O-dimethoxytrityl-λ/-acyl-nucleosides 16a or 16b or 16c or 3'-O-(4- azidobutyryl)-5'-O-dimethoxytritylthymidine 16d. Procedures for derivatization were described in Examples 4-6. The resulting dry solid phases contained 0.03-0.08 mmol of DMTr-groups per gram of solid support (8). All four nucleoside-bound solid supports 17a-d (Figure 4) performed well in standard oligonucleotide synthesis.

B. Derivatization of nanoparticles with oligonucleotide

Example 8

Sigma-Aldrich 3 -Aminopropyl- functionalized silica nanoparticles, 3% (w/v) in ethanol (average particle size = 15 nm), 2.5 ml were evaporated to dryness and re-suspended in dioxane (1.9 ml).

A solution of 10 μmol of azidoalkyl-tethered oligonucleotide 18 (Figure 5) in aqueous triethylammonium hydrogen carbonate (2 M, 0.05 ml) and triphenylphosphine (0.03 g) were added to the suspension of nanoparticles and the mixture was periodically gently shaken for 48 h at room temperature. The supernatant was removed by centrifugation. The nanoparticles were re-suspended in dioxane and the supernatant was removed by centrifugation (2 times), re-suspended in 40% aqueous ethanol and the supernatant was removed by centrifugation (2 times). The resulting oligonucleotide 18 functionalized nanoparticles (Figure 6) were re-suspended in a buffer containing 10 mM Tris-HCl, pH 7.0; 0.1 M NaCl; 10 mM MgCl ₂ to give 3% (w/v) and analyzed.

Fluorescein-labeled oligonucleotide 19 (Figure 5) (oligonucleotide 19 had a sequence complementary to oligonucleotide 18, 1 μmol in 0.1 ml of buffer containing 10 mM Tris-HCl, pH 7.0; 0.1 M NaCl; 10 mM MgCl ₂ ) was added to a suspension of oligonucleotide 18 functionalized nanoparticles (0.1 ml). The mixture was gently shaken for 1 h at room temperature. The supernatant was removed by centrifugation, nanoparticles were re-suspended in 40% aqueous ethanol and the supernatant was removed by centrifugation (3 times). The resulting particles were re-suspended in 80% aqueous ethanol (3% w/v). These nanoparticles were of intense green color.

Fluorescein-labeled oligonucleotide 20 (Figure 5) (oligonucleotide 20 had a sequence non-complementary to oligonucleotide 18, 1 μmol in 0.1 ml of water) was added to a suspension of oligonucleotide 18 functionalized nanoparticles (0.1 ml). The mixture was gently shaken for 1 h at room temperature. The supernatant was removed by centrifugation, nanoparticles were re-suspended in a buffer containing 10 mM Tris- HCl, pH 7.0; 0.1 M NaCl; 10 mM MgCl ₂ and the supernatant was removed by

centrifugation (3 times). The resulting particles were re-suspended in 80% aqueous ethanol (3% w/v). These nanoparticles were of extremely pale green color.

C. Functionalization of microarray surface with oligonucleotide

Example 9

Two Amine-derivatized slides (Erie Scientific Company) were immersed in a mixture of dioxane (1.88 ml) and aqueous triethylammonium hydrogen carbonate (2M, 0.05 ml), containing azidoalkyl-tethered oligonucleotide 18 (Figure 5), 10 μmol. Tri- phenylphosphine (30 mg) was added and the slides were gently shaken for 48 h at room temperature. The oligonucleotide 18 functionalized slides (Figure 7) were then washed with 50% aqueous ethanol (2 times), water (2 times), dioxane (2 times), ethanol (2 times) and dried.

A solution of fluorescein-labeled oligonucleotide 19 (Figure 5) (oligonucleotide 19 had a sequence complementary to oligonucleotide 18, 1 μmol in 0.1 ml of buffer containing 10 mM Tris-HCl, pH 7.0; 0.1 M NaCl; 10 mM MgCl ₂ ) was manually spotted on the first oligonucleotide 18 functionalized slide. The slide was gently shaken for 1 h at room temperature. The slide was washed with buffer containing 10 mM Tris- HCl, pH 7.0; 0.1 M NaCl; 10 mM MgCl ₂ (3 times) water, followed by ethanol and finally dried. The resulting slide had several intense green color spots.

When oligonucleotide 20 (Figure 5) (oligonucleotide 20 had a sequence non- complementary to oligonucleotide 18) was spotted on the second slide and subsequently washed as described for the first slide, the resulting second slide had several visible spots of extremely pale green color.

The present invention is not limited in scope by specified embodiments described herein. All additional modifications of the invention described herein and resulting from description and figures will appear apparent to those skilled in the art. All such modifications are falling within the scope of claims appended herein.

The disclosures of various cited patents and publications are incorporated herein by reference and are not falling within the scope of the claims appended herein.

References

1. International patent application No. WO 2005/061445 (Langstrom et al, "Methods for carbon isotope labeling synthesis by rhodium-promoted carbonylation via isocyanate using azides and carbon-isotope monoxide")

2. George, W. The ICI Polyuretanes Book, Ed2, 1990, Published jointly by ICI and John Wiley & Sons, N.Y.

3. Paul, F. Coordination Chemistry Reviews, 2000, 203, 269-323.

4. Valli, V. L. K., Alper, H. J. Org. Chem. 1995, 60, 257-258.

5. Braverman, S., Cherkinsky, M., Kedrova, L., Reiselman, A. Tetrahedron Letters, 1999, 40, 3235-3238.

6. Azhayev, A., Antopolsky, M. Tetrahedron, 2001, 57, 4977-4986.

7. Azhayev, A., Antopolsky, M. U.S. Patent No. 6,770,754 and European Patent Application No. 1 404 695.

8. Atkinson, T., Smith, M. in Oligonucleotide Synthesis. A Practical Approach; Gait, M.J. Ed.; IRL Press: Oxford, 1984, p. 111.

Scheme 1

R'-XH compound of P(III) Ia-d R-N ₃ ► R-N=C=O R-NH-C-X-R'

TTa-d ^ trialkylammonium Il hydrogen carbonate buffer IIIa-d IVa-d O

where R = an organic group and

R' = an aliphatic organic group, X = NH or NR' '(Ia and IVa), where R' ' = alkyl;

R' = an aliphatic organic group, X = S (Ib and IVb);

R' = an aromatic organic group, X = S (Ic and IVc);

R' = an aromatic organic group, X = O (Id and IVd);

The applications of this reaction are shown as follows:

R-N ₃ →→ R-NH-CO-NR'R' ' Synthesis of urea derivatives

R-N ₃ →→ R-NH-CO-SR' Synthesis of thiocarbamate derivatives

R-N ₃ →→ R-NH-CO-OR' Synthesis of carbamate derivatives

Scheme 2