Chemoselective thiol-conjugation with alkene or alkyne-phosphonothiolates and - phosphonates

Process for the preparation of a phosphonothiolate or phosphonate comprising the step of:

• Reacting a compound of formula (I*)
• wherein
• V represents C1-C8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl;
• X represents (R3 R4)C;
• wherein , Y, R1, R3 and R4 are as defined in any one of the preceding claims,
• with a thiol-containing molecule of formula (II)
• wherein is as defined in any one of the preceding claims;
• resulting in a compound of formula (III*)
• wherein , , V, R1, X and Y are as defined in any one of the preceding claims.

The process according to any one of the preceding claims, wherein R1 represents C1-C8-alkyl optionally substituted with at least one of (C1-C8-alkoxy)n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, Br, I, —N2, —N(C1-C8-alkyl)H, —NH2, —N3, —N(C1-C8-alkyl)2, ═O, C3-C8-cycloalkyl, —S—S—(C1-C8-alkyl), hydroxy-(C1-C8-alkoxy)n wherein n is 1, 2, 3, 4, 5 or 6, C2-C8-alkenyl, C2-C8-alkynyl;

• or
• R1 represents phenyl optionally independently substituted with at least one of C1-C8-alkyl, (C1-C8-alkoxy)n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, I, Br, —NO2, —N(C1-C8-alkyl)H, —NH2 or —N(C1-C8-alkyl)2;
• or
• R1 represents a 5- or 6-membered heteroaromatic system such as pyridyl;
• preferably, R1 represents C1-C8-alkyl, C1-C8-alkyl substituted with —S—S—(C1-C8-alkyl), C1-C8-alkyl substituted with (C1-C8-alkoxy)n wherein n is 1, 2, 3, 4, 5 or 6, C1-C8-alkyl substituted with optionally substituted phenyl;
• or phenyl;
• or phenyl substituted with —NO2.

The process according to any one of the preceding claims, wherein represents a small molecule such as, for example, an optionally substituted C1-C8-alkyl, preferably ethyl, —CH2-phenyl,

• wherein # indicates the position of the Y;
• or
• represents an optionally substituted phenyl;
• or
• represents a radioactive or non-radioactive nuclide, biotin, a reporter enzyme, a nucleotide, an oligonucleotide, a fluorophore such as CY5 or EDANS, an amino acid, a peptide, or an optionally substituted 5- or 6-membered heteroaromatic system;
• wherein optionally the further comprises a linker that is bound to the Y.

Process for the preparation of a compound of formula (I) or formula (I*), said method comprising:

• (I) reacting a compound of formula (Ia)
• of formula (I*a)
• wherein
• L represents a linker suitable for binding to an amino acid, a peptide, a nucleotide or an oligonucleotide;
• and
• , X, Y, V and R1 are as defined in any one of the preceding claims,
• with an amino acid, a peptide, a nucleotide or an oligonucleotide, wherein the amino acid, the peptide, the nucleotide or the oligonucleotide is bound to a solid support,
• resulting in a compound of formula (Ib) or (I*b),
• wherein Z represents the amino acid, the peptide, the nucleotide or the oligonucleotide, wherein the amino acid, the peptide, the nucleotide or the oligonucleotide is bound to the solid support;
• and
• (II) cleaving the compound of formula (Ib) or formula (I*b) from the solid support to give the compound of formula (I) or formula (I*):
• wherein represents the amino acid, the peptide, the nucleotide or the oligonucleotide bound to the Y through the linker L, and
• , X, Y, V and R1 are as defined above.

A compound of formula (III)

• wherein
• represents a bond and X represents (R3 R4)C, wherein R3 and R4 independently represent H or C1-C8-alkyl;
• or
• represents a double bond and X represents R3C, wherein R3 represents H or C1-C8-alkyl;
• and
• , , R1, and Y are as defined in any one of the preceding claims.

A kit comprising a solid support, and

• a compound of formula (Ia)
• and/or a compound of formula (I*a)
• wherein L is a linker suitable for binding to an amino acid, a peptide, a nucleotide or an oligonucleotide;
• and
• wherein , R1, X, Y, and V are as defined in any one of the preceding claims.

A compound of formula (IIIa)

• wherein
• represents a bond and X represents (R3 R4)C;
• or
• represents a double bond and X represents R3—C;
• and
• , , R1, X and Y are as defined in any one of the preceding claims.