US2020231539A1PendingUtilityA1
Multiple cycloaddition reactions for labeling of molecules
Assignee: EUROPEAN MOLECULAR BIOLOGY LABORATORYPriority: Jan 14, 2014Filed: Nov 22, 2019Published: Jul 23, 2020
Est. expiryJan 14, 2034(~7.5 yrs left)· nominal 20-yr term from priority
G01N 33/533G01N 33/582C07C 271/22C07C 271/34
56
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Claims
Abstract
The present invention relates to methods for linking tetrazines with dienophiles to establish at least two linkages by sequentially performing at least two cycloaddition reactions. The methods in particular allow establishing multi-labeling strategies. In particular, the invention relates to methods for forming linkages by cycloaddition reactions, wherein the method comprises reacting a first alkyl-substituted tetrazine with a first dienophile comprising a irans-cyclooctenyl group followed by reacting a second tetrazine with a second dienophile comprising a cyclooctynyl group, wherein the reaction of the first tetrazine with the first dienophile proceeds in the presence of the second dienophile.
Claims
exact text as granted — not AI-modified1 . A method for forming linkages by cycloaddition reactions, wherein the method comprises reacting a first tetrazine with a first dienophile followed by reacting a second tetrazine with a second dienophile, wherein the reaction of the first tetrazine with the first dienophile proceeds in the presence of the second dienophile, wherein
(i) the first tetrazine comprises a group of the formula:
wherein
R 3 is C 1 -C 3 -alkyl;
(ii) the first dienophile comprises a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
(iii) the second tetrazine comprises a group of the formula:
(iv) the second dienophile comprises a cyclooctynyl group of the formula:
wherein
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl.
2 . The method of claim 1 , wherein the first tetrazine preferentially reacts with the first dienophile in the presence of the second dienophile.
3 . The method of claim 1 , wherein the rate constant k of the first tetrazine with the first dienophile is usually at least 10 2 -times higher than the rate constant k of the reaction of the first tetrazine with the second dienophile.
4 . The method of claim 1 , wherein the rate constant k of the first tetrazine with the first dienophile is allowed to proceed for 30 minutes or less at a temperature of about 37° C.
5 . The method of claim 1 , wherein the method comprises contacting a target molecule or a target molecule composition with
(i) a first labeling agent comprising a group of the formula:
wherein
R 3 is C 1 -C 3 -alkyl; followed by
(ii) a second labeling agent comprising a group of the formula:
wherein the target molecule comprises
(i) a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl; and
(ii) a cyclooctynyl group of the formula:
wherein
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl,
wherein the target molecule composition comprises
(i) a first target molecule comprising a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl; and
(ii) a second target molecule comprising a cyclooctynyl group of the formula:
wherein
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl.
6 . The method of claim 1 , wherein the trans-cyclooctenyl group has the formula:
wherein
R 1 is hydrogen;
the first tetrazine or labeling agent comprises a group of the formula:
wherein
R 3 is methyl;
the cyclooctynyl group has the formula:
wherein
R 2 is hydrogen; and
the second tetrazine or labeling agent comprises a group of the formula:
7 . The method of claim 1 , wherein the first dienophile or the target molecule or target molecule composition is reacted with the first tetrazine or labeling agent under conditions that allow for substantially all trans-cyclooctenyl groups to react prior to reacting the second dienophile or the target molecule or target molecule composition with the second tetrazine or labeling agent.
8 . A cell comprising
(i) a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
and
(ii) a cyclooctynyl group of the formula:
wherein
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl.
9 . The cell of claim 8 , wherein the cyclooctynyl group is attached to a first polypeptide and the trans-cyclooctenyl group is attached to a second polypeptide, the first and the second polypeptide being different polypeptides.
10 . A method for preparing the cell of claim 8 , which comprises
a) providing a cell comprising:
(i) a first aminoacyl tRNA synthetase, or a polynucleotide encoding it; and optionally a second aminoacyl tRNA synthetase, or a polynucleotide encoding it;
(ii) a first tRNA having an anticodon to a first selector codon, or a polynucleotide encoding said tRNA; and optionally a second tRNA having an anticodon to a second selector codon, or a polynucleotide encoding said tRNA; and
(iii) a polynucleotide encoding a target polypeptide and comprising one or more than one first and second selector codon(s); or a first polynucleotide encoding a first target polypeptide and comprising one or more than one first selector codon(s) and a second polynucleotide encoding a second target polypeptide and comprising one or more than one second selector codon(s),
wherein said first aminoacyl tRNA synthetase (i) is capable of acylating the first tRNA (ii) with a first unnatural amino acid or an analogue thereof comprising a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl,
and with a second unnatural amino acid or an analogue thereof comprising a cyclooctynyl group of the formula:
wherein
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
or
wherein said first aminoacyl tRNA synthetase (i) is capable of acylating the first tRNA (ii) with a first unnatural amino acid or an analogue thereof comprising a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl, and
said second aminoacyl tRNA synthetase (i) is capable of acylating the second tRNA (ii) with a second unnatural amino acid or an analogue thereof comprising a cyclooctynyl group of the formula:
wherein
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
b) contacting the cell with the first and the second unnatural amino acid or an analogue thereof; and
c) allowing translation of the polynucleotide(s) (iii) thereby incorporating the first and the second unnatural amino acids or the analogues thereof into the target polypeptide(s) at the position(s) encoded by the selector codon(s).
11 . The method of claim 10 , wherein the first unnatural amino acid or the analogue thereof is a compound of the formula:
wherein:
X 1 has the formula:
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
X 2 is —CH 2 —, —O—, —S—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NH—C(O)— or —C(O)—NH—;
X 3 is C 1 -C 6 -alkylene, —(CH 2 —CH 2 —O) m —, —(CH 2 —O) p — or a single bond;
X 4 is —NH—, —C(O)—NH—, —NH—C(O)—, —NH—CH(NH 2 )—, —CH(NH 2 )—NH—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH 2 )—, —C(O)—NH—C(NH)—NH—, NH—CH(NH 2 )—C(O)— or —NH—C(NH)—NH—C(O)—;
X 5 is —(CH 2 ) n — or phenylene-CH 2 —;
X 6 is hydrogen, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy-C 1 -C 2 -alkyl, C 2 -C 7 -alkanoyloxy-C 1 -C 2 -alkyl or C 2 -C 7 -alkanoylsulfanyl-C 1 -C 2 -alkyl;
R 4 is —OH or —NH 2 ;
n is an integer from 1 to 4;
m is an integer from 1 to 6; and
p is an integer from 1 to 6,
or an acid or base addition salt thereof, and
wherein the second unnatural amino acid or the analogue thereof is a compound of the formula:
wherein:
X 1 has the formula:
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
X 2 is —CH 2 —, —O—, —S—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NH—C(O)— or —C(O)—NH—;
X 3 is C 1 -C 6 -alkylene, —(CH 2 —CH 2 —O) m —, —(CH 2 —O) p — or a single bond;
X 4 is —NH—, —C(O)—NH—, —NH—C(O)—, —NH—CH(NH 2 )—, —CH(NH 2 )—NH—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH 2 )—, —C(O)—NH—C(NH)—NH—, NH—CH(NH 2 )—C(O)— or —NH—C(NH)—NH—C(O)—;
X 5 is —(CH 2 ) n — or phenylene-CH 2 —;
X 6 is hydrogen, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy-C 1 -C 2 -alkyl, C 2 -C 7 -alkanoyloxy-C 1 -C 2 -alkyl or C 2 -C 7 -alkanoylsulfanyl-C 1 -C 2 -alkyl;
R 4 is —OH or —NH 2 ;
n is an integer from 1 to 4;
m is an integer from 1 to 6; and
p is an integer from 1 to 6,
or an acid or base addition salt thereof.
12 . A polypeptide comprising
(i) a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
and
(ii) a cyclooctynyl group of the formula:
wherein
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl.
13 . The polypeptide of claim 12 , comprising a first residue of the formula:
wherein:
X 1 has the formula
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
X 2 is —CH 2 —, —O—, —S—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NH—C(O)— or —C(O)—NH—;
X 3 is C 1 -C 6 -alkylene, —(CH 2 —CH 2 —O) m —, —(CH 2 —O) p — or a single bond;
X 4 is —NH—, —C(O)—NH—, —NH—C(O)—, —NH—CH(NH 2 )—, —CH(NH 2 )—NH—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH 2 )—, —C(O)—NH—C(NH)—NH—, NH—CH(NH 2 )—C(O)— or —NH—C(NH)—NH—C(O)—;
X 5 is —(CH 2 ) n — or phenylene-CH 2 —;
Z 1 is —O— or NH—;
n is an integer from 1 to 4;
m is an integer from 1 to 6; and
p is an integer from 1 to 6,
and a second residue of the formula:
wherein:
X 1 has the formula:
R 2 is hydrogen, halogen, C 1 -C 4 -alkyl, (R c O) 2 P(O)O—C 1 -C 4 -alkyl, (R d O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino;
R c , R d independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl;
X 2 is —CH 2 —, —O—, —S—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NH—C(O)— or —C(O)—NH—;
X 3 is C 1 -C 6 -alkylene, —(CH 2 —CH 2 —O) m —, —(CH 2 —O) p — or a single bond;
X 4 is —NH—, —C(O)—NH—, —NH—C(O)—, —NH—CH(NH 2 )—, —CH(NH 2 )—NH—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH 2 )—, —C(O)—NH—C(NH)—NH—, NH—CH(NH 2 )—C(O)— or —NH—C(NH)—NH—C(O)—;
X 5 is —(CH 2 ) n — or phenylene-CH 2 —;
Z 1 is —O— or NH—;
n is an integer from 1 to 4;
m is an integer from 1 to 6; and
p is an integer from 1 to 6.
14 . An unnatural amino acid comprising a trans-cyclooctenyl group of the formula:
wherein
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, Di-(C 1 -C 4 -alkyl)amino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl,
or an analogue of the unnatural amino acid.
15 . The unnatural amino acid of claim 14 , having the formula:
wherein
X 1 is a trans-cyclooctenyl group of the formula:
R 1 is hydrogen, halogen, C 1 -C 4 -alkyl, (R a O) 2 P(O)O—C 1 -C 4 -alkyl, (R b O) 2 P(O)—C 1 -C 4 -alkyl, CF 3 , CN, hydroxyl, C 1 -C 4 -alkoxy, —O—CF 3 , C 2 -C 5 -alkenoxy, C 2 -C 5 -alkanoyloxy, C 1 -C 4 -alkylaminocarbonyloxy or C 1 -C 4 -alkylthio, C 1 -C 4 -alkylamino, C 2 -C 5 -alkenylamino, C 2 -C 5 -alkenyl-C 1 -C 4 -alkyl-amino or Di-(C 2 -C 5 -alkenyl)amino; and
R a , R b independently are hydrogen or C 2 -C 5 -alkanoyloxymethyl.
X 2 is —CH 2 —, —O—, —S—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NH—C(O)— or —C(O)—NH—;
X 3 is C 1 -C 6 -alkylene, —(CH 2 —CH 2 —O) m —, —(CH 2 —O) p —, or a single bond;
X 4 is —NH—, —C(O)—NH—, —NH—C(O)—, —NH—CH(NH 2 )—, —CH(NH 2 )—NH—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH 2 )—, —C(O)—NH—C(NH)—NH—, NH—CH(NH 2 )—C(O)— or —NH—C(NH)—NH—C(O)—;
X 5 is —(CH 2 ) n — or phenylene-CH 2 —;
X 6 is hydrogen, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy-C 1 -C 2 -alkyl, C 2 -C 7 -alkanoyloxy-C 1 -C 2 -alkyl or C 2 -C 7 -alkanoylsulfanyl-C 1 -C 2 -alkyl;
R 4 is —OH or —NH 2 ;
n is an integer from 0 to 4;
m is an integer from 1 to 6; and
p is an integer from 1 to 6,
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