Target sequences for synthetic molecules

1 . A vector comprising a nucleic acid sequence encoding a bonding partner, wherein the nucleic acid sequence encoding the bonding partner comprises a nucleic acid sequence encoding a carrier polypeptide and a nucleic acid sequence encoding a target sequence, wherein the nucleic acid sequence encoding the target sequence is heterologous to the nucleic acid sequence encoding the carrier polypeptide, and the target sequence comprises at least 2 cysteines and has the sequence of Cys-Cys-R n , whether R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the target sequence reacts with a biarsenical molecule having the formula:

and tautomers, anhydrides, and salts thereof;

wherein:

each X 1 or X 2 is independently Cl, Br, I, OR a , or SR a , or

X 1 and X 2 together with the arsenic atom form a ring having the formula:

R a is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;

Y 1 and Y 2 , are each independently H or CH 3 , or

Y 1 and Y 2 together form a ring such that the biarsenical molecule has the formula

wherein:

M is O, S, CH 2 , C(CH 3 ) 2 , or NH;

R 1 and R 2 are each independently OR a , OAc, NR a R b , or H;

R 3 and R 4 are each independently H, F, Cl, Br, I, OR a , or R a ; or

R 1 together with R 3 , or R 2 together with R 4 , or both, form a ring in which

(i) one of R 1 or R 3 is C 2 -C 3 alkyl and the other is NR a and

(ii) one of R 2 and R 4 is C 2 -C 3 alkyl and the other is NR a ;

R b is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:

wherein the spiro linkage is formed at C 1

2 . The vector of claim 1 , wherein the target sequence is Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2 (SEQ. ID NO: 3), wherein:

X 1 is an amino acid having a non-polar side chain,

X 2 is an amino acid having a basic side chain, and

X 3 is an amino acid having a non-ionic polar side chain.

3 . The vector of claim 2 , wherein X 1 is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.

4 . The vector of claim 2 , wherein X 2 is lysine, arginine, or histidine.

5 . The vector of claim 2 , wherein X 3 is asparagine, glutamine, serine, or threonine.

6 . The vector of claim 1 , wherein the target sequence is Cys-Cys-gly-gly-lys-gly-asn-gly-gly-Cys-gly-Cys-his (SEQ. ID NO: 50).

7 . The vector of claim 1 , wherein the nucleic acid sequence encoding the target sequence is attached at the 5′-end of the nucleic acid sequence encoding the carrier polypeptide.

8 . The vector of claim 1 , wherein the nucleic acid sequence encoding the target sequence is attached at the 3′-end of the nucleic acid sequence encoding the carrier polypeptide.

9 . The vector of claim 1 , wherein the biarsenical molecule has the following structure:

10 . A method of labeling a carrier molecule, comprising:

a) providing a bonding partner comprising the carrier molecule and a target sequence, and

b) contacting the bonding partner with a biarsenical molecule under conditions wherein the biarsenical molecule reacts with the target sequence, wherein the target sequence comprises at least 2 cysteines and has the sequence Cys-Cys-R n , wherein R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the biarsenical molecule has the formula:

and tautomers, anhydrides, and salts thereof;

wherein:

each X 1 or X 2 is independently Cl, Br, I, OR a , or SR a , or

X 1 and X 2 together with the arsenic atom form a ring having the formula:

R a is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;

Y 1 and Y 2 are each independently H or CH 3 , or

Y 1 and Y 2 together form a ring such that the biarsenical molecule has the formula

wherein:

M is O, S, CH 2 , C(CH 3 ) 2 , or NH;

R 1 and R 2 are each independently OR a , OAc, NR a R b , or H;

R 3 and R 4 are each independently H, F, Cl, Br, I, OR a , or R a ; or

R 1 together with R 3 , or R 2 together with R 4 , or both, form a ring in which

(i) one of R 1 or R 3 is C 2 -C 3 alkyl and the other is NR a and

(ii) one of R 2 and R 4 is C 2 -C 3 alkyl and the other is NR a ;

R b is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:

wherein the spiro linkage is formed at C 1 .

11 . The method of claim 10 , wherein the biarsenical molecule generates a detectable signal.

12 . The method of claim 11 , further comprising monitoring the detectable signal.

13 . The method of claim 11 , wherein the signal is a fluorescent signal.

14 . The method of claim 10 , wherein the biarsenical molecule is coupled to a solid phase.

15 . The method of claim 10 , wherein the target sequence is coupled to a solid phase.

16 . The method of claim 10 , wherein the carrier molecule is a polypeptide.

17 . The method of claim 16 , wherein the polypeptide is an antibody or an enzyme.

18 . The method of claim 10 , wherein the target sequence is Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2 (SEQ. ID NO: 3), wherein:

X 1 is an amino acid having a non-polar side chain,

X 2 is an amino acid having a basic side chain, and

X 3 is an amino acid having a non-ionic polar side chain.

19 . The method of claim 18 , wherein X 1 is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.

20 . The method of claim 18 , wherein X 2 is lysine, arginine, or histidine.

21 . The method of claim 18 , wherein X 3 is asparagine, glutamine, serine, or threonine.

22 . The method of claim 10 , wherein the target sequence is Cys-Cys-gly-gly-lys-gly-asn-gly-gly-Cys-gly-Cys-his (SEQ. ID NO: 50).

23 . A kit comprising:

a) a biarsenical molecule having the structure:

and tautomers, anhydrides, and salts thereof;

wherein:

each X 1 or X 2 is independently Cl, Br, I, OR a , or SR a , or

X 1 and X 2 together with the arsenic atom form a ring having the formula:

R a is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;

Y 1 and Y 2 are each independently H or CH 3 , or

Y 1 and Y 2 together form a ring such that the biarsenical molecule has the formula

wherein:

M is O, S, CH 2 , C(CH 3 ) 2 , or NH;

R 1 and R 2 are each independently OR a , OAc, NR a R b , or H;

R 3 and R 4 are each independently H, F, Cl, Br, I, OR a , or R a ; or

R 1 together with R 3 , or R 2 together with R 4 , or both, form a ring in which

(i) one of R 1 or R 3 is C 2 -C 3 alkyl and the other is NR a and

(ii) one of R 2 and R 4 is C 2 -C 3 alkyl and the other is NR a ;

R b is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:

wherein the spiro linkage is formed at C 1 ; and

b) a bonding partner comprising a target sequence, wherein the target sequence comprises at least 2 cysteines and has the sequence Cys-Cys-R n , wherein R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the target sequence reacts with the biarsenical molecule.

24 . The kit of claim 23 , wherein the target sequence is Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2 (SEQ. ID NO: 3), wherein:

X 1 is an amino acid having a non-polar side chain,

X 2 is an amino acid having a basic side chain, and

X 3 is an amino acid having a non-ionic polar side chain.

25 . The kit of claim 24 , wherein X 1 is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.

26 . The kit of claim 24 , wherein X 2 is lysine, arginine, or histidine.

27 . The kit of claim 24 , wherein X 3 is asparagine, glutamine, serine, or threonine.

28 . The kit of claim 23 , wherein the target sequence is Cys-Cys-gly-gly-lys-gly-asn-gly-gly-Cys-gly-Cys-his (SEQ. ID NO: 50).

29 . A method of purifying a bonding partner, comprising:

a) providing a bonding partner comprising a target sequence, comprises at least 2 cysteines and has the sequence of Cys-Cys-R n , whether R is any amino acid and n is an integer from 1-100, and wherein when n≧2, each R is selected independently from every other R in the sequence, but wherein the target sequence cannot be Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) wherein Xaa can be any amino acid, and wherein the target sequence reacts with a biarsenical molecule having the structure:

and tautomers, anhydrides, and salts thereof;

wherein:

each X 1 or X 2 is independently Cl, Br, I, OR a , or SR a , or

X 1 and X 2 together with the arsenic atom form a ring having the formula:

R a is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Z is 1,2-ethanediyl, 1,2-propanediyl, 2,3-butanediyl, 1,3-propanediyl, 1,2 benzenediyl, 4-methyl-1,2-benzenediyl, 1,2-cyclopentanediyl, 1,2-cyclohexanediyl, 3-hydroxy-1,2-propanediyl, 3-sulfo-1,2-propanediyl, or 1,2-bis(carboxy)-1,2-ethanediyl;

Y 1 and Y 2 are each independently H or CH 3 , or

Y 1 and Y 2 together form a ring such that the biarsenical molecule has the formula

wherein:

M is O, S, CH 2 , C(CH 3 ) 2 , or NH;

R 1 and R 2 are each independently OR a , OAc, NR a R b , or H;

R 3 and R 4 are each independently H, F, Cl, Br, I, OR a , or R a ; or

R 1 together with R 3 , or R 2 together with R 4 , or both, form a ring in which

(i) one of R 1 or R 3 is C 2 -C 3 alkyl and the other is NR a and

(ii) one of R 2 and R 4 is C 2 -C 3 alkyl and the other is NR a ;

R b is H, C 1 -C 4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;

Q is CR a R b , CR a OR b , C═O, or a spirolactone having the formula:

wherein the spiro linkage is formed at C 1 ;

d) contacting the bonding partner with the biarsenical molecule, wherein the biarsenical molecule is coupled to a solid phase,

e) eluting the bonding partner from the biarsenical molecule by contacting the biarsenical molecule with a dithiol.

30 . The method of claim 29 , wherein the carrier molecule is a protein.

31 . The method of claim 30 , wherein the protein is an antibody or an enzyme.

32 . The method of claim 29 , wherein the biarsenical compound is membrane permeable.

33 . The method of claim 29 , wherein the dithiol is selected from the group consisting of 1,2-benzendithiol, 1,2-cyclohexanedithiol and 1,2-ethanedithiol.

34 . An isolated polypeptide comprising a sequence Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2 (SEQ. ID NO: 3), wherein:

X 1 is an amino acid having a non-polar side chain,

X 2 is an amino acid having a basic side chain, and

X 3 is an amino acid having a non-ionic polar side chain.

35 . The polypeptide of claim 34 , wherein X 1 is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.

36 . The polypeptide of claim 34 , wherein X 2 is lysine, arginine, or histidine.

37 . The polypeptide of claim 34 , wherein X 3 is asparagine, glutamine, serine, or threonine.

38 . An isolated polypeptide comprising a sequence Cys-Cys-Gly-Gly-Lys-Gly-Asn-Gly-Gly-Cys-Gly-Cys-His (SEQ. ID. NO: 50).

39 . An isolated nucleic acid molecule encoding a polypeptide having a sequence Cys-Cys-X 1 -X 1 -X 2 -X 1 -X 3 -X 1 -X 1 -Cys-X 1 -Cys-X 2 (SEQ. ID NO: 3), wherein:

X 1 is an amino acid having a non-polar side chain,

X 2 is an amino acid having a basic side chain, and

X 3 is an amino acid having a non-ionic polar side chain.

40 . The isolated nucleic acid molecule of claim 39 , wherein X 1 is glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan.

41 . The isolated nucleic acid molecule of claim 39 , wherein X 2 is lysine, arginine, or histidine.

42 . The isolated nucleic acid molecule of claim 39 , wherein X 3 is asparagine, glutamine, serine, or threonine.

43 . An isolated nucleic acid molecule encoding a polypeptide having a sequence Cys-Cys-Gly-Gly-Lys-Gly-Asn-Gly-Gly-Cys-Gly-Cys-His (SEQ. ID. NO: 50).