LU503039B1 - Novel photocleavable spin-labels - Google Patents
Novel photocleavable spin-labels Download PDFInfo
- Publication number
- LU503039B1 LU503039B1 LU503039A LU503039A LU503039B1 LU 503039 B1 LU503039 B1 LU 503039B1 LU 503039 A LU503039 A LU 503039A LU 503039 A LU503039 A LU 503039A LU 503039 B1 LU503039 B1 LU 503039B1
- Authority
- LU
- Luxembourg
- Prior art keywords
- alkyl
- alkylene
- formula
- group
- bond
- Prior art date
Links
- 230000005298 paramagnetic effect Effects 0.000 claims abstract description 80
- 125000000217 alkyl group Chemical group 0.000 claims description 399
- 125000001424 substituent group Chemical group 0.000 claims description 93
- 150000001875 compounds Chemical class 0.000 claims description 63
- 125000004432 carbon atom Chemical group C* 0.000 claims description 51
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 46
- 229910021481 rutherfordium Inorganic materials 0.000 claims description 34
- 239000002243 precursor Substances 0.000 claims description 32
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 30
- 229910052702 rhenium Inorganic materials 0.000 claims description 29
- 239000000126 substance Substances 0.000 claims description 28
- 238000005481 NMR spectroscopy Methods 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 24
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 22
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 21
- 238000005259 measurement Methods 0.000 claims description 21
- -1 nitroxides Chemical class 0.000 claims description 21
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 18
- 229910052703 rhodium Inorganic materials 0.000 claims description 18
- 229910021645 metal ion Inorganic materials 0.000 claims description 17
- 229910052757 nitrogen Inorganic materials 0.000 claims description 17
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 16
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 14
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 14
- 125000002947 alkylene group Chemical group 0.000 claims description 13
- 125000004076 pyridyl group Chemical group 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- 230000015556 catabolic process Effects 0.000 claims description 11
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims description 10
- 125000006526 (C1-C2) alkyl group Chemical group 0.000 claims description 8
- 238000004435 EPR spectroscopy Methods 0.000 claims description 8
- 125000006193 alkinyl group Chemical group 0.000 claims description 8
- 239000002738 chelating agent Substances 0.000 claims description 8
- 229910052760 oxygen Inorganic materials 0.000 claims description 8
- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 6
- 229910052717 sulfur Inorganic materials 0.000 claims description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 5
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- 125000006704 (C5-C6) cycloalkyl group Chemical group 0.000 claims description 3
- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims description 3
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- 238000002360 preparation method Methods 0.000 abstract description 2
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- 239000000243 solution Substances 0.000 description 41
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- 238000001228 spectrum Methods 0.000 description 22
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- 239000012286 potassium permanganate Substances 0.000 description 18
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 16
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- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
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- 239000007832 Na2SO4 Substances 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
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- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 125000002653 sulfanylmethyl group Chemical group [H]SC([H])([H])[*] 0.000 description 1
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/44—Iso-indoles; Hydrogenated iso-indoles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
- C07F7/1804—Compounds having Si-O-C linkages
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/10—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using electron paramagnetic resonance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/28—Details of apparatus provided for in groups G01R33/44 - G01R33/64
- G01R33/281—Means for the use of in vitro contrast agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
- G01R33/46—NMR spectroscopy
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Plural Heterocyclic Compounds (AREA)
Abstract
The present invention refers to light induced removable paramagnetic tags, their preparation and uses.
Description
New Luxembourg Patent Application
Applicants: Silva et al.
Our ref.: AVS17949LU
LU503039
Novel photocleavable spin-labels
In order to be able to develop new medicines, it is necessary to understand the molecular basics of a disease. It is important to understand the processes that underlie a disease and to understand how they are regulated.
Spin-labeled molecules have a wide range of applications in materials science and spectroscopy. A spin- labeled molecule is an organic molecule that possesses an unpaired electron (paramagnetic) and has the ability to bind to another molecule.
As an example, Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) has received great attention in the past decades and has found applications in various areas of science, such as drug discovery and structural biology. PRE-NMR is used to unveil the three-dimensional structure of individual biomolecules, as well as megadalton protein and protein—nucleic acids complexes. In (PRE)-
NMR spectroscopy, the tagged-molecule displays a magnetic dipolar interaction between the NMR- active nuclei and the unpaired electron(s) of the paramagnetic center(s), which results in an increase in nuclear relaxation rates. This effect is distance-dependent and gives information about the three- dimensional structure of the studied molecule and its mode of interaction with partners, as well as interdomain dynamics. Differently, in Electron Paramagnetic Resonance (EPR), structure information can be gained by measuring the distance between two or more spin labels. In most cases, the molecule under investigation does not contain paramagnetic centers, which therefore need to be introduced by site- directed spin labelling. For this, the spin-label must contain the unpaired electron and a specific reactive group (usually alkyl halides, terminal alkynes, azides, Michael-acceptors and others) to form a covalent bond with the molecule. The most widely used spin-labels are nitroxides (also called nitroxyl radicals), due to their great stability, and metal-chelators complexes that bind a paramagnetic cation. The spin labels need to be stable, have, as far as possible, a rigid structure and should react easily with the desired site.
It could be observed that all the spin labels have a reactive group (halide, carboxylic acid, Michael acceptor, among others) which allow them to be attached to a desired molecule. For example, a spin label 1s connected to an amino acid or a phosphoramidite building block in order to incorporate the spin-label 1 by means of solid phase peptide synthesis and/or RNA/DNA synthesizer, respectively. Given all these requirements, the development of spin labels 1s an area of increasing interest.
However, as an example, after 3-(2-iodoacetamido)-PROXYL (CAS number 27048-01-7) is attached to a molecule, it (the paramagnetic tag) cannot be easily removed. Upon addition of a reducing agent, the tag changes from the paramagnetic to the diamagnetic state by the formation of the corresponding hydroxylamine group, but the molecule of interest does not recover its native state (as prior to the spin- label attachment).
A first aspect of the present invention refers to a paramagnetic compound for formula (I)
PT-PG-RG (I) wherein
PT is a chemical system comprising at least one unpaired electron which system is connected with PG via one or two bonds, preferably a system selected from the group consisting of nitroxides, chelating agents;
RG is a reactive group selected from the group consisting of -(C0-C5)alkylene-halide (preferably -(CO-C5)alkylene-I, -(CO-C5)alkylene-Br, -(C0-C5)alkylene-Cl); -(CO-C5)alkylene-OTs (i.e. -O- tosyl, 1e- 0O-S02-CsHs-CH3); -(CO-C5)alkylene-O-SO2-(C1-C5)alkyl (preferably -(CO-
C5)alkylene-OMs (i.e. -(CO-C5)alkylene-O-mesyl, i.e. -(CO-C5)alkylene-O-SO2-CHz); -(CO-
C5)alkylene-O-C(O)-phenyl; -(CO-C5)alkylene-S-SO2-(C1-C5)alkyl; -(CO-C5)alkylene-OH; - (CO-C5)alkylene-NHz; -(C0-C5)alkylene-NH(C1-C5)alkyl; a terminal alkinyl or azide function, (e.g., -(CO-C5)alkylene-N3; -O-(C1-C5)alkylene-N3; -NH-(C1-C5)alkylene-N3;-O-C(O)-(CO-
C5)alkylene-N3; -N((CO-C5)alkyl)-C(O)-(C1-C5)alkylene-N3 (preferably -NH-C(O)-(C1-
C5)alkylene-N3), -O-(C1-C5)alkylene-ethinyl; -(CO-C5)alkylene-ethinyl; N)(CO-CS)alky1)-(C1- 2
C5)alkylene-ethinyl (preferably -NH-(C1-C5)alkylene-ethinyl); -O-C(O)-(CO-C5)alkylene- ethinyl; -N((C0-C5)alkyl)-C(O)-(C0-C5)alkylene-ethinyl | (preferably = -NH-C(O)-(CO-
C5)alkylene-ethinyl); Michael acceptor residues such as imidyl, preferably maleimidyl, acrylamidyls, acrylates, and -(C0-C5)alkylene-S-S-pyridinyl (the term encompasses ortho, meta and para positions of the S-bond to the nitrogen of the pyridine ring) wherein the pyridine ring may be optionally substituted independently from each other with one, two, three or four (C1-
C5)alkyl; and
PGs a photocleavable group selected from the group consisting of
A) formula (IT)
R2
R4 R1—=
R5 NO,
R3 (IT) wherein -* indicates the bond to RG;
R1 is either -(CO-C5)alkylene-C(O)-*, -(CO-C5)alkylene-C(O)O-(CO-C5)alkylene-*, -(C1-
C5)alkylene-OC(O)-(CO-C5)alkylene-*, -(C1-C5)N(CO-C5)alky1)(C(O)-(CO-C5)alkylene-* and -(C1-C5)O(C(O)-(CO-C5)alkylene-* or -(C1-C5)-alkylene-*,
R2, R3, R4 and RS independently from each other represent -H, -(CO-C5)alkylene-halide, (preferably -(CO-CS)alkylene-I, -(CO-C5)alkylene-Br, -(CO-C5)alkylene-CI, more preferably -I (Le. -(C0)alkylene-I), -Cl or -Br, even more preferably -Br or -CI), -(C1-C5)-alkyl, -(CO-
C5)alkylene-O-(C1-C5)-alkyl, -C(O)O(CO-CS)alkyl) (preferably COOH), -C(O)O-(C1-C5)- 3 alkyl, -(C0-C5)alkylene-N((CO0-C5)alkyl)((CO-C5)alkyl) (preferably -N((CO-CS5)alkyl)((CO-
C5)alkyl), i.e. -(C0-C5)alkylene is -CO-alkylene), a direct bond to PT (-*), -C(O)NH-*, -O-*, -(CO-
C5)alkylene-ethinylene-(CO-C5)alkylene-*. {C1-C5)alkylene-*, -N((C0-C5)alkyl)H-C(O)-(CO-
C5)alkyl*, -C(0)0-*, -O-C(O)-, -C(O)NH-CH2-CH2-NHC(O)-*, -(C0-C5)alkylene-N(C1-
C5)alkyl-* or
A,
Oy # wherein ” indicates the bond between PG and PT; provided that either one of R2, R3, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)) or comprises a bond to PT (-*) (preferably a bond to PT of formula (VII), (VII), (IX), (IX), or (X)), preferably, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VII), (IX), (IX’), or (X)) or comprises a bond to PT (preferably a bond to PT of formula (VID), (VII), (IX), (IX), or (X)); or
R4 and R5 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R4 and RS are direct bonds to PT(-*) (preferably a
PT of formula (V) or (VI)) , and R2 and R3 independently from each other represent -H, -(C1-
C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl), preferably R2 and R3 independently from each other represent -H, - (C1-C5)-alkyl,
R2 and R4 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R2 and R4 are direct bonds to PT (-*) (preferably 4
PT of formula (V) or (VI)) , and RS and R3 independently from each other represent -H, -(C1-
C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl), preferably RS and R3 independently from each other represent -H, - (C1-C5)-alkyl,
R3 and R5 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R3 and R5 are direct bonds to PT (-*) (preferably
PT of formula (V) or (VI) , and R2 and R4 independently from each other represent -H, -(C1-
C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl), preferably RS and R3 independently from each other represent -H, - (C1-C5)-alkyl,;
In one preferred embodiment, R1 is selected from the group consisting of -(CO-C5S)alkylene-
C(O)-*, -(CO-C5)alkylene-C(O)O-(CO-CS)alkylene-*, -(CO-C5)alkylene-OC(O)-(CO-
C5)alkylene-*, and -(C1-C5)-alkylene-*, more preferred selected from the group consisting of -
C(O)-*, -(CO-C5)alkylene-C(O)O-(CO-CS)alkylene-*, -(CO-C5)alkylene-OC(O)-(CO-
C5)alkylene-*, and (C1-C2)-alkylene; or even more preferably R1 is (C1-C2)-alkylene); or
B) formula (III) *
R7
R4 R9 =
R5 O R6
R8 (II) wherein
-* indicates the bond to RG;
R4 and RS are as defined in A);
R6 represents O or S;
R7 and R8 independently from each other represent -H, -(CO-C5)alkylene-halide, (preferably - (CO-CS)alkylene-I, -(CO-C5)alkylene-Br, -(CO-C5)alkylene-CI, more preferably -I (Le. (CO)alkylene-I), - Cl or -Br, even more preferably -Br or -CI), -(C1-C5)-alkyl, -(CO-C5)alkylene-
O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-CS)alky1)((CO-
C5)alkyl), a direct bond to PT (-*), -C(O)NH-, -O-*, -(C0-C5)alkylene-ethinylene-(CO-
C5)alkylene-*. (C1-C5)alkylene-*, -N((CO-CS)alkyl)H-C(O)-(CO-CS5)alkyl*, -C(0)O-*, -OC(O)- # -C(O)NH-CH2-CH2-NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl- or
A,
Cy # wherein * indicates the bond between PG and PT; and
RO represents -(C1-C5)alkyl or -H; provided that either one of R4, RS, R7 or R8 is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), 1X’), or (X)) or comprises a bond to PT (-) (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)), preferably, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VIID), (IX), (IX), or (X)) or comprises a bond to PT (preferably a PT of formula (VII), (VIII), (IX), 1X’), or (X));
Preferably, R7 or R8 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)- alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(C0-C5)alkylene-N((C0-C5)alkyl)((CO-C5)alkyl)); or 6
R4 and R5 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (-*) (preferably, R4 and R5 are direct bonds to PT (preferably of formula (V) or (VI)) , and R7 and R8 independently from each other represent -H, -(C1-C5)- alkyl, -O-(Cl1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-
C5)alky1)((CO-C5)alkyl); or
R7 and R4 each independently form a direct bond to PT (-*) (preferably a PT of formula (V) or (VD) or comprise a direct bond to said PT (-*) (preferably, R7 and R4 are direct bonds to PT (preferably of formula (V) or (VD)) , and RS and R8 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-CS)alkylene-N((CO-
CS)alkyl)((CO-C5)alkyl); or
R8 and R5 each independently form a direct bond to PT (-) (preferably a PT of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R5 and R8 are direct bonds to PT (preferably of formula (V) or (VD)) , and R4 and R7 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-CS)alkylene-N((CO-
CS)alky1l)((CO-CS)alky1); or
C) formula (IV) *
R14 R12
R15 =
NV R13
R16 O R6
R17 (IV) wherein 7
-* indicates the bond to RG;
R6 and R12 independently from each other represent O or S;
R14, R15, R16 and R17 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-
C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-CS)alky1)((CO-CS)alky1); and
R13 is a direct bond to PT (-), -O-*, -(CO-C5)alkylene-ethinylene-(C0-C5)alkylene-*. —(C1-
C5)alkylene-*, -(C0-C5)alkylene-N(C0-C5)alkyl-* or
A,
Qu # wherein * indicates the bond between PG and PT.
In one preferred embodiment of aspect one or any of its other preferred embodiments, PT in the paramagnetic compound is elected from the group consisting of nitroxides, chelating agents.
In another preferred embodiment of aspect one or any of its other preferred embodiments, PG is selected from the group consisting of formula (II) or formula (III); and
PT is selected from the group consisting of formula (V)
Rg
Rh Rf
RA,
N +
Pt 07 X
Ra Rb 8
(V) wherein “+” indicates the positions of R4 and RS, or R2 and R4, or R3 and RS, respectively, in formula (IT); or the positions of R4 and R5, or R7 and R4, or R8 and RS, respectively in formula (IIT), respectively, and
Rf and Rg independently from each other represent -H, -(C1-C5)alkyl, -(C5-C6)cycloalkyl, -
C(O)OH, -C(O)NH2, -OH, -NH2, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl;
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ra or Rb is a -(C5-C6)cycloalkyl, the other substituent is -(C1-C5)alkyl); or
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (Cl-
C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (Cl-
C5)alkylene-OH;
Ri and Rh independently from each other represent (C1-C5)alkyl, (C5-C6)cycloalkyl or phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH; or 9 formula (VI) which is attached to PG via two bonds (see definitions of R2 to RS in formula (IT); and definitions of R4 to R8 in formula (III), respectively):
Rk
Rj ++ +
N
“À .O
Ra Rb (VI) wherein “+” indicates the position of R4 and R5, or R2 and R4, or R3 and RS, respectively, in formula (IT); or the positions of R4 and RS, or R7 and R4, or R8 and RS, respectively in formula (II), respectively, and
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH;
Rj and Rk independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Rj or Rk is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Rj and Rk form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and
(C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1 -
C5)alkylene-OH; or formula (VIT) which 1s attached to PG via one bond
Reine
Ry RE
Rio” Rea 07 X ke ° Ha R&B (VII) wherein + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (IIT), respectively; and
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH;
Ri and Rh independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or 11
Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH;
Rc, Rd, and Rg independently from each other represent -H, -(C1-C5)alkyl, -(CS-
C6)cycloalkyl, -C(O)OH, -C(O)NHz, -OH, -NHb, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; and
Rf and Re, respectively, either independently from each other represent -(C1-C5)alkyl, -(CO-
C5)alkylene-N((CO-C5)alkyl)((CO-CS)alkyl), -(CO-C5)alkylene-COO(CO-CS)alkyl or -H; or Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and
Re are attached to; or formula (VIII)
Rf +
R Re
Rh Rb
Ri | Ra
O
(VII) wherein + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; and wherein 12
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH;
Ri and Rh independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH;
Rg represents -H, -(C1-C5)alkyl, -(C5-C6)cycloalkyl, -C(O)OH; -C(O)NHz, -OH, -NHz, or - pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl;
Rf and Re, independently from each other represent (C1-C5)alkyl or H; or Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to: or formula (IX) 13
© OH
HO Da
TN
O
+
Me A
N O oH
O OH
(IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of
Gd(III), Mn(II), Yb(III), Fe(II), Co(II), Dy(IID), Ce(III), Tm(IID); and wherein + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; or formula (IX)
O
HO T
NW
O
Me
N O
04 À
O OH
(ax) (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(III), Mn(IT), Yb(IIT), Fe(II), Co(II), Dy(IID), Ce(III), Tm(III); and 14 wherein + indicates the position of any one of R2 to R5 of formula (II); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; or formula (X)
RI +
NN
N N
C Me ) „N N
Rm \__/ Rn (X) wherein
Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT),
Mn(ID), Yb(IIT), Fe(II), Co(II), Dy (III), Ce(III), Tm(IID); and wherein + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; and
RI, Rm and Rn are independently from each other selected from the group consisting of -H, - (C1-C5)alkyl, -(C1-CS)alkylene-C(O)O(C0-CS)alkyl, -(C1-CS)alkylene-C(O)N((CO-
C5)alkyl)2, -(C1-C5)alkylene-N((CO-C5)alky1)2, (C1-C5)alkylene-S-SO2-(C1-C5)alkyl, and - (C1-C5)alkylene-C(O)NH-(C1-C5)alkylene-S-SO2-(C1-C5)alkyl; and + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively.
In one preferred embodiment of aspect one or any of its other preferred embodiments, PG is of formula (IV); and PT is selected from the group consisting of formula (VII), (VIII), (IX), (IX) or (X) wherein + indicates the position of R13 of formula (IV) and all definitions are those as described in claim 3.
In one preferred embodiment of aspect one or any of its other preferred embodiments, PG is of formula (ID)
R2
R4 R1—=
R5 NO,
R3 (I) wherein R1 to RS and -* are as defined in claim 1.
Another aspect refers to a precursor of a paramagnetic compound according to aspect one or any of its preferred embodiments, wherein the precursor is of formula (I)’
PT’-PG-RG
Formula (I)’ wherein PG and RG are as defined in any one of the preceding claims and PT” is of formula (IX), (IX) or (X), respectively, but in which the Me is not present.
Another preferred embodiment of aspect one or any of its other preferred embodiments or aspect two or any of its preferred embodiment refers to a paramagnetic compound or precursor, respectively, wherein
PT is of formula (VII) or (VIII). 16
Another preferred embodiment of aspect one or any of its other preferred embodiments or aspect two or any of its preferred embodiment refers to a paramagnetic compound or precursor, respectively, wherein
RG is selected from the group consisting of -I, -Br, -CI, -O-tosyl and -O-mesyl, -S-SO2-(CHs), -S-S- pyridine, -OH, -NHz, -NH(C1-C2)alkyl, and -maleimideyl.
Another preferred embodiment of aspect one or any of its other preferred embodiments refers to a paramagnetic compound, wherein PG is selected from the group consisting of formula (II); and R1 in formula (IT) is -CH2-.
Another preferred embodiment of aspect one or any of its other preferred embodiments refers to a paramagnetic compound, wherein PG is selected from the group consisting of formula (IT) and (III); wherein R1 in formula (IT) is -CHz-; and PT is selected from the group consisting of formula (VII) or formula (VIII), wherein Ra, Rb, Rh and Ri each represent methyl and Rc to Rg in formula (VII) each represent -H and Re to Rg in formula (VIII) each represent -H, respectively.
Another aspect (three) refers to a conjugate of formula (XI)
R2
R4 R1-x— target molecule
R5 NO,
R3 (XI) wherein R1-RS5 are as defined in any of the preceding claims; and
X represents -S-¥, -O-% -NH-*, -S-S-% -O-C(O) -*, -NH-C(0)-N((C0-C5)alkyl) -¥, -NH-C(O) +, 0 0 ~ N oy X Ar \ 0
N° $ „N N ' LA ; § , | , OF © . wherein -® indicates the bond to a carbon atom of the target molecule (TM); 17 or a conjugate of formula ((XIT)
TM
X
R7
R4 R9
R5 O R6
R8 (XII) wherein R4-R9 are as defined in any one of the preceding claims; and
X represents -S-%, -0-3, -NH-*, -S-S-3, -O-C(O) -%, -NH-C(O)-N((CO-C5)alkyl) =, -NH-C(O) -,
O
0 ~ N
Mr N 28 x
NT $ „N N
LS. , 0 § , Or wherein -* indicates the bond to a carbon atom of the target molecule (TM); or a conjugate of formula ((XIII)
TM
X
R14 R12
R15 =
NV R13
R16 O R6
R17 (XII) 18 wherein R6 and R12-R17 are as defined in any one of the preceding claims; and
X represents -S-¥, -O-S, -NH-*, -S-S-$, -O-C(O) -*, -NH-C(O)-N((CO-C5)alkyl) -¥, -NH-C(O) +,
KK 0
A 28 \ O
Y ° N 2H. $ § , | „or © wherein -% indicates the bond to a carbon atom of the target molecule (TM).
Another aspect (four) refers to a method to analyze a target molecule comprising the steps of 1) reacting the target molecule with at least one molecule of formula (I) resulting in a conjugate; or reacting the target molecule with at least one precursor molecule of formula (I)’ and then adding a Me as defined herein to prepare the PT in the conjugate; 2) performing at least one measurement selected from the group consisting of a Paramagnetic
Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement, an electron spin resonance (ESR) measurement, and a fluorescence resonance energy transfer microscopy measurement with the conjugate; 3) removing the paramagnetic tag by exposing the conjugate to light of a wave length which is suitable to break down the PG in the conjugate, preferably exposing the conjugate to light of a wave length between 200 nm and 620 nm, more preferably between 220 nm and 600 nm; 4) optionally recovering the target molecule in its native state (native state = structure of the target molecule before it was reacted with at least one molecule of formula (I)); or recovering the target molecule in an altered form in that the reactive group of the target molecule with which the RG of a molecule of formula (I) reacted, is changed after the breakdown of a PG.
Another aspect (five) refers to a method to analyze a target molecule comprising the steps of 1) reacting the target molecule with at least two different molecules of formula (I), wherein the at least two different molecules differ in that the PG of each molecule breaks down at a different 19 wavelength/in a different wavelength range, resulting in a conjugate with at least two different
PG groups; or reacting the target molecule with at least two different molecules of formula (I) or formula (XI), respectively, wherein the at least two different molecules differ in that the PG of each molecule breaks down at a different wavelength/in a different wavelength range which are both within the range between 200 nm and 620 nm, more preferably between 220 nm and 600 nm, resulting in a conjugate with at least two different PG groups; if at least one compound of formula (XI) is present, add a Me as defined herein to prepare the PT in the conjugate; 2) performing a Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement with the conjugate with at least two different PG groups; 3) removing one paramagnetic tag by exposing the conjugate with at least two different PG groups to light of a wavelength which is suitable to break down one PG group while the other PG group is not affected by said wavelength; 4) performing a Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement with the conjugate resulting from step 3); 5) removing a further paramagnetic tag by exposing the conjugate after step 4) to light of a wavelength which is suitable to break down a second PG group which was not affected by the wavelength of step 3); optionally recovering the target molecule in its native state (native state = structure of the target molecule before it was reacted with at least one molecule of formula (I)); or recovering the target molecule in an altered form in that the reactive group of the target molecule with which the RG of a molecule of formula (I) reacted, is changed after the breakdown of at least one of the two PGs.
Another aspect (six) refers to a kit comprising a compound according to aspect one or any of its preferred embodiments or a precursor of aspect two or any of its preferred embodiments in a separated container.
In one prefere dembodiment of aspect six, the kit further comprising a manual instructing the user how to react a compound of formula (I) or a precursor of formula (I)’ with a target molecule resulting in a conjugate or a precursor conjugate.
The term “(Cn-Cm)” in front of a chemical residue, wherein m 1s greater than n and both are natural numbers between 0 and 5 (1.e., 0, 1, 2, 3, 4, or 5), indicates the number of C-atoms of a group. In case n is 0, a group is not present, i.e. the term refers to hydrogen in case of a terminal group (e.g. (C1)alkyl = -CHz and (C0)-alkyl = -H) or to a bond in case of a bridging group, e.g. an alkylene group, ((C1)alkylene = -CHz- and (CO)alkylene = -). For clarification sake, the term (C1-C2)alkyl means the group can be either -CHs (methyl) or -CH2-CHs (ethyl) and the term (C1-C2)alkylene means the group can be either -
CHz- (methylene) or -CH2-CHz- (ethylene).
The term “alkyl” as used herein refers to a chemical group of the general formula ChHa.n in case n is 3 or more, the bond of the residue to the core structure can be in form of a primary, secondary or tertiary bond.
The term “alkylene” as used herein refers to a chemical bridging group of the general formula (CnH2n)-.
In case Nis 2 or more, the two bridging bonds can be either at one carbon atom or at two different carbon atoms of the group. For example: (C2)alkylene can be either
H LH H H
AX H
H or .
In the terms “-N((C0-C5)alkyl)((C0-C5)alkyl)” and “-N((CO-CS)alky1l)2”, respectively, and also in any other term with two or more (Cn-Cm)groups, it is understood that each (Cn-Cm)group can be selected individually. E.g., the term -N((CO-C5)alkyl)2 can refer to group -NHCHz or -N(CHs)2 or -NHz or -
N(CH3)(C2Hs5) etc.
The term “paramagnetic tag” (abbreviation “PT”) as used herein refers to a molecular system comprising an unpaired electron. The molecular system is connected via one or two bonds with a photocleavable group according to the invention. 21
The term “photocleavable group” as use herein refers to a chemical group that can undergo a cleavage after interacting with light of a certain wavelength in the range between 200 nm and 620 nm. Various classes of groups/compounds can be used as a PG for the compounds according to the invention, preferably groups according to A) to C) below.
The term R “comprise a bond to PT” means - for all structures of compounds according to the invention - a respective R is selected from the group consisting of -C(O)NH-*, -O-*, -(CO-C5)alkylene- ethinylene-(C0-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-C5)alkyl)H-C(O)-(C0-C5)alky1*, -C(0)O-*, -0C(0)-*, -C(O)NH-CHz-CH»-NHC(O)-*, -(C0-C5)alkylene-N(C1-C5)alkyl-* or
A, er # wherein ” indicates the bond between PG and PT)
For clarification’s sake, the term “one of R2 to R5” means only one of R2, R3, R4 or RS is or comprises a bond to PT.
The skilled person is aware that all embodiments disclosed herein, irrespectively if they are named “embodiments”, “preferred embodiments”, “more preferred embodiments” etc. can be combined with each other as long as such a combination does not contradict a law of nature. For example, an embodiment, wherein PT is of formula (V) cannot be combined with an embodiment, wherein PT is of formula (VI) because PT can only be either of the two structures; but an embodiment, wherein PT is of formula (V) can be combined with any other embodiment, wherein, e.g., PG has a preferred structure.
The present invention refers to new compounds of formula (I) that contain a paramagnetic tag (PT) and a photocleavable group (PG) in their chemical structures which can be attached to a target molecule via 22 specific reactive groups (RG). Such new compounds of formula (I) can be selectively photo-cleaved, resulting in essentially complete removal of the spin-label (paramagnetic tag) upon light irradiation.
Spin-labelling defines the process of attaching a paramagnetic tag (PT), to specific positions of target molecules via a photocleavable group (PG) and a reactive group (RG).
This gives the possibility to “tag” a PT to a molecule and liberate (“un-tag”) this functionality with light irradiation. Light is a clean energy source, can be applied selectively at a well-defined wavelength and can be delivered to the sample in a controlled manner through LEDs or LASERs. The skilled person will understand that the various PGs need different wavelengths to undergo a cleavage.
Depending on the functionalization of the chromophore, the photochemical and photophysical properties can be fine-tuned and the wavelength of the absorbed light can be controlled. Accordingly, if two or more paramagnetic tags containing PPGs that absorb light of different wavelengths can be introduced in a molecule in a site-specific manner, the individual tags could be released selectively and successively, by applying the corresponding wavelength. This procedure would offer the opportunity of studying the effect of multiple paramagnetic tags using only one sample: the tags would be released from the molecule one by one, using light of different wavelength, thus allowing to investigate the effect of each individual tag. In the end, the molecule could be recovered in its native form and used for other experiments. This procedure is particularly promising to study protein complexes, where different tags can be introduced at cysteine located on different molecules before complex assembly.
Thus, one aspect of the present invention refers to a paramagnetic compound of formula (I)
PT-PG-RG (I) wherein
PT is a chemical system comprising at least one unpaired electron, preferably one unpaired electron, which system is connected with PG via one or two bonds, preferably a system selected from the group consisting of nitroxides, chelating agents; 23
RG is a reactive group selected from the group consisting of -(CO-C5)alkylene-halide (preferably -(CO-
C5)alkylene-I, -(CO-C5)alkylene-Br, -(CO-CS)alkylene-CI); -(CO-C5)alkylene-OTs (i.e. -O-tosyl, i.e.-
O-SO2-C6H4-CH:); -(CO-C5)alkylene-O-SO2-(C1-C5)alkyl (preferably -(CO-C5)alkylene-OMS (i.e. - (CO-CS5)alkylene-O-mesyl, i.e. -(C0-C5)alkylene-O-SO2-CHs); -(CO-C5)alkylene-O-C(O)-phenyl; - (CO-C5)alkylene-S-SO2-(C1-C5)alkyl; -(CO-C5)alkylene-OH; -(CO-C5)alkylene-NHz; -(CO-
C5)alkylene-NH(C1-C5)alkyl; a terminal alkinyl or azide function, (e.g., -(CO-C5)alkylene-N3; -O- (C1-C5)alkylene-N3; -NH-(C1-C5)alkylene-N3;-O-C(O)-(CO-C5)alkylene-N3; -N((C0-C5)alkyl)-C(O)- (C1-C5)alkylene-N3 (preferably -NH-C(O)-(C1-C5)alkylene-N3), -O-(C1-C5)alkylene-ethinyl; -(CO-
C5)alkylene-ethinyl; N)(CO-C5)alkyl)-(C1-C5)alkylene-ethinyl (preferably -NH-(C1-C5)alkylene- ethinyl); -O-C(O)-(CO-CS)alkylene- ethinyl; -N((C0-C5)alkyl)-C(O)-(C0-C5)alkylene-ethinyl (preferably -NH-C(O)-(CO-C5)alkylene-ethinyl); Michael acceptor residues such as imidyl, preferably maleimidyl, acrylamidyls, acrylates, and -(CO-CS)alkylene-S-S-pyridinyl (the term encompasses ortho, meta and para positions of the S-bond to the nitrogen of the pyridine ring) wherein the pyridine ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; and
PG is a photocleavable group selected from the group consisting of
A) formula (IT)
R2
R4 R1—=
R5 NO,
R3 (In wherein -* indicates the bond to RG; 24
RI is either -(CO-C5)alkylene-C(O)-*, -(CO-C5)alkylene-C(O)O-(CO-C5)alkylene-*, -(C1-
C5)alkylene-OC(O)-(CO-C5)alkylene-*, -(C1-C5)N(CO-C5)alky1)(C(O)-(CO-CS)alkylene-* and -(C1-
C5)O(C(O)-(CO-CS)alkylene-* or -(C1-C5)-alkylene-*,
R2, R3, R4 and RS independently from each other represent -H, -(C0-C5)alkylene-halide, (preferably -(CO-C5)alkylene-I, -(CO-C5)alkylene-Br, -(CO-C5)alkylene-CI, more preferably -I (i.e. - (C0)alkylene-I), -CI or -Br, even more preferably -Br or -CI), -(C1-C5)-alkyl, -(CO-CS)alkylene-O- (C1-C5)-alkyl, -C(O)O(CO-CS)alkyl) (preferably COOH), -C(O)O-(C1-C5)-alkyl, -(CO-CS)alkylene-
N((CO-CS)alkyl)((CO-C5)alkyl) (preferably -N((CO-CS)alky1)((CO-C5)alkyl), i.e. -(CO-CS)alkylene is -
CO-alkylene), a direct bond to PT (-"), -C(O)NH-*, -O-*, -(C0-C5)alkylene-ethinylene-(CO-
C5)alkylene-*. —(C1-C5)alkylene-*, -N((CO-C5)alkyl)H-C(O)-(CO-CS5)alkyl*, -C(0)O-*, -O-C(0)-*, -
C(O)NH-CHz-CHz-NHC(O)-*, -(CO-C5)alkylene-N(C1-CS)alky1-" or
A,
Qi # wherein * indicates the bond between PG and PT; provided that either one of R2, R3, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)) or comprises a bond to PT (=) (preferably a bond to PT of formula (VII), (VIII), (IX), (IX), or (X)), preferably, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)) or comprises a bond to PT (preferably a bond to PT of formula (VII), (VIII), (IX), (IX), or (X)); or
R4 and RS each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R4 and R5 are direct bonds to PT(-") (preferably a PT of formula (V) or (VI)) , and R2 and R3 independently from each other represent -H, -(C1-C5)-alkyl, -O- (C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-CS)alkyl)((CO-CS)alkyl), preferably R2 and R3 independently from each other represent -H, -(C1-C5)-alkyl;
R2 and R4 each independently form a direct bond to PT (preferably of formula (V) or (VD)) or comprise a direct bond to said PT (preferably, R2 and R4 are direct bonds to PT (-*) (preferably PT of formula (V) or (VI)) , and RS and R3 independently from each other represent -H, -(C1-C5)-alkyl, -O- (C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-CS)alkyl)((CO-CS)alkyl), preferably RS and R3 independently from each other represent -H, -(C1-C5)-alkyl;
R3 and RS each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R3 and RS are direct bonds to PT (-*) (preferably PT of formula (V) or (VI)) , and R2 and R4 independently from each other represent -H, -(C1-C5)-alkyl, -O- (C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-CS)alkyl)((CO-CS)alkyl), preferably RS and R3 independently from each other represent -H, -(C1-C5)-alkyl;
In one preferred embodiment, R1 is selected from the group consisting of -(CO-C5)alkylene-C(O)-*, - (CO-C5)alkylene-C(O)O-(C0-C5)alkylene-*, -(CO-C5)alkylene-OC(O)-(CO-C5)alkylene-*, and -(C1-
C5)-alkylene-*, more preferred selected from the group consisting of -C(O)-*, -(CO-C5)alkylene-
C(O)O-(CO-CS)alkylene-*, -(CO-C5)alkylene-OC(O)-(CO-C5)alkylene-*, and (C1-C2)-alkylene; or even more preferably R1 is (C1-C2)-alkylene); or
B) formula (III) *
R7
R4 R9
SN
RS O R6
R8 26
(II) wherein -* indicates the bond to RG;
R4 and RS are as defined in A);
R6 represents O or S;
R7 and R8 independently from each other represent -H, -(C0-C5)alkylene-halide, (preferably -(CO-
C5)alkylene-I, -(C0-CS5)alkylene-Br, -(CO-CS)alkylene-CI, more preferably -I (i.e. (CO)alkylene-I), - CI or -Br, even more preferably -Br or -Cl), -(C1-C5)-alkyl, -(C0-C5)alkylene-O-(C1-C5)-alkyl, -
C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-CS)alkylene-N((CO-CS)alky1)((CO-CS)alkyl), a direct bond to
PT (-%), -C(O)NH-*, -O-*, -(C0-C5)alkylene-ethinylene-(C0-C5)alkylene-*. (C1-C5)alkylene-*, -
N((C0-C5)alkyl)H-C(0)-(C0-C5)alkyl®, -C(O)O-*, -OC(0)-*, -C(O)NH-CH2-CH>-NHC(0)-*, -(CO-
C5)alkylene-N(C1-C5)alkyl-" or
A nd # wherein * indicates the bond between PG and PT; and
RO represents -(C1-C5)alkyl or -H; provided that either one of R4, R5, R7 or R8 is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)) or comprises a bond to PT (-") (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)), preferably, R4 or R5 is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)) or comprises a bond to PT (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)); 27
Preferably, R7 or R8 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)-alkyl, -
C(O)OH, -C(0)0-(C1-C5)-alkyl, -(CO-CS)alkylene-N((CO-CS)alkyl)((CO-CS)alky1l)); or
R4 and RS each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (-*) (preferably, R4 and R5 are direct bonds to PT (preferably of formula (V) or (VI)) , and R7 and R8 independently from each other represent -H, -(C1-C5)-alkyl, -O- (C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-CS)alkylene-N((CO-CS)alky1)((CO-CS)alkyl); or
R7 and R4 each independently form a direct bond to PT (-*) (preferably a PT of formula (V) or (VI)) or comprise a direct bond to said PT (-*) (preferably, R7 and R4 are direct bonds to PT (preferably of formula (V) or (VI)) , and RS and R8 independently from each other represent -H, -(C1-C5)-alkyl, -O- (C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-CS)alkylene-N((CO-CS)alky1)((CO-CS)alkyl); or
R8 and RS each independently form a direct bond to PT (-*) (preferably a PT of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, RS and R8 are direct bonds to PT (preferably of formula (V) or (VI)), and R4 and R7 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)- alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(C0-C5)alkylene-N((CO-C5)alkyl)((CO-C5)alkyl); or
C) formula (IV) *
R14 R12
R15 >
NV R13
R16 O R6
R17 (IV) 28 wherein -* indicates the bond to RG;
R6 and R12 independently from each other represent O or S;
R14, R15, R16 and R17 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)- alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-CS5)alkylene-N((CO-CS)alky1)((CO-CS)alky1l); and
R13 is a direct bond to PT (-*), -O-*, -(CO-C5)alkylene-ethinylene-(C0-C5)alkylene-*. -(C1-
C5)alkylene-*, -(C0-C5)alkylene-N(C0-C5)alkyl- or
A N
Cp # wherein * indicates the bond between PG and PT.
PT (paramagnetic tag)
In one preferred embodiment, PT is selected from the group consisting of (1) formula (V) which is attached to PG via two bonds (see definition of R4 and RS in formula (IT) and (IIT), respectively):
Rg
Rh Rf
Ri AK +
N + ~~ „07 VW
Ra Rb 29
(V) wherein “+” indicates the positions of R4 and RS, or R2 and R4, or R3 and RS, respectively, in formula (ID); or the positions of R4 and RS, or R7 and R4, or R8 and RS, respectively in formula (III), respectively, and
Rf and Rg independently from each other represent -H, -(C1-C5)alkyl, -(C5-C6)cycloalkyl, -C(O)OH, -
C(O)NHz, -OH, -NHz, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl;
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ra or Rb is a -(C5-C6)cycloalkyl, the other substituent is -(C1-C5)alkyl); or
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH;
Ri and Rh independently from each other represent (C1-C5)alkyl, (C5-C6)cycloalkyl or phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; (2) formula (VI) which is attached to PG via two bonds (see definitions of R2 to RS in formula (ID); and definitions of R4 to R8 in formula (III), respectively):
Rk
Rj ++
N +
À
.O
Ra Rb (VI) wherein “+” indicates the position of R4 and R5, or R2 and R4, or R3 and RS, respectively, in formula (ID); or the positions of R4 and RS, or R7 and R4, or R8 and RS, respectively in formula (III), respectively, and
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH;
Rj and Rk independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Rj or Rk is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Rj and Rk form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; (e.g.: a preferred structure, wherein R4 and R5 each form a direct bond to PT is 31
;
O-N
NO,
X
(3) formula (VII) which is attached to PG via one bond
Riese
Ri RE = (Re © Re ° Ha %b (VID) wherein + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III), respectively; and
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH;
Ri and Rh independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or 32
Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH;
Rc, Rd, and Rg independently from each other represent -H, -(C1-C5)alkyl, -(C5-C6)cycloalkyl, -
C(O)OH, -C(O)NHz, -OH, -NHz, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; and
Rf and Re, respectively, either independently from each other represent -(C1-C5)alkyl, -(CO-
C5)alkylene-N((CO-CS)alkyl)((CO-C5)alkyl), -(CO-C5)alkylene-COO(CO-CS)alkyl or -H; or Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to; and (4) formula (VIII)
Rf +
R Re
Rh Rb
Ri | Ra
O
(VII) wherein + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; and wherein
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or 33
Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH;
Ri and Rh independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or
Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-
OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH;
Rg represents -H, -(C1-C5)alkyl, -(C5-C6)cycloalkyl, -C(O)OH; -C(O)NHz, -OH, -NHz, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl;
Rf and Re, independently from each other represent (C1-C5)alkyl or H; or Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to; and (5) formula (IX) 34
© OH
HO Da
TN
O
+
Me A
N O oH
O OH
(IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT),
Mn(II), Yb(III), Fe(II), Co(II), Dy (III), Ce(III), Tm(III); and wherein + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; or (6) formula (IX)
O
HO T
NW
O
Me
N O
04 À
O OH
(IX) (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of
Gd(IIT), Mn(ID), Yb(III), Fe(II), Co(II), Dy(III), Ce(III), Tm(III); and wherein + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; or (7) formula (X)
RI +
NN
N N
C Me ) „N N
Rm \__/ Rn (X) wherein
Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT), Mn(Il),
Yb(III), Fe(II), Co(II), Dy(III), Ce(III), Tm(IID); and wherein + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; and
RI, Rm and Rn are independently from each other selected from the group consisting of -H, -(C1-
C5)alkyl, -(C1-C5)alkylene-C(O)O(CO-CS)alkyl, -(C1-CS)alkylene-C(O)N((CO-CS)alky1)2, -(C1-
C5)alkylene-N((CO-C5)alky1)2, -(C1-C5)alkylene-S-SO2-(C1-C5)alkyl, and -(C1-C5)alkylene-
C(O)NH-(C1-C5)alkylene-S-SO2-(C1-C5)alkyl; and + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively.
Preferred embodiments 36
RG
In one preferred embodiment, RG is a reactive group selected from the group consisting of -I; -Br; -CI; -OTs (i.e. -O-tosyl, i.e. -O-SO2-C6H4-CH:); -OMs (i.e. -mesyl, i.e. -O-SO2-CHs); -O-C(O)-phenyl; -S-
SO2-(C1-C5)alkyl; -OH; -NHz; -NH(C1-C5)alkyl; a terminal azide function selected from the group consisting of -(CO-C5)alkylene-N3, -O-(C1-C5)alkylene-N3, -NH-(C1-C5)alkylene-Ns, -O-C(O)-(C1-
C5)alkylene-N3, and -NH-C(O)-(C1-C5)alkylene-N3; a terminal alkinyl function selected from the group consisting of -O-(C1-C5)alkylene-ethinyl; -(CO-C5)alkylene-ethinyl; -NH-(C1-C5)alkylene- ethinyl; -O-C(O)-(CO-C5)alkylene-ethinyl; -NH-C(O)-(C0-C5)alkylene-ethinyl; -maleimidyl, - acrylamidyl, -acrylates, and -S-S-pyridinyl, wherein the pyridine ring may be optionally substituted independently from each other with one, two, three or four (C1-C2)alkyl.
In another preferred embodiment, a reactive group RG is selected from the group consisting of —(CO-
C5)alkylene-I, —(CO-C5)alkylene-Br, —(CO0-C5)alkylene-Cl, —(CO0-C5)alkylene-O-tosyl and —(CO-
C5)alkylene-O-SO2-(C0-C5(alkyl), -S-SO2-(CHs), -S-S-pyridinyl wherein the pyridine ring may be optionally substituted with one, two, three or four substituents independently from each other selected from the group consisting of -(C1-C2)alkyl, -((CO-C5)alkylene)-OH, -((CO-C5)alkylen)-NHz, -((CO-
C5)alkylen)-NH(C1-C5)alkyl, and Michael acceptors selected from the group consisting of imide, acrylamide, acrylate.
In yet another preferred embodiment, a reactive group RG is selected from the group consisting of -I, -
Br, -CI, -O-tosyl and -O-mesyl, -S-SO2-(CHz), -S-S-pyridine, -OH, -NH2, -NH(C1-C2)alkyl, and - maleimidyl. -I, -Br, -CI, -tosyl and -O-SOz-(C0-CS(alkyl)
In one preferred embodiment, a reactive group RG is selected from the group consisting of —(CO-
C5)alkylene-I, —(CO-C5)alkylene-Br, —(CO-C5)alkylene-CI, —(CO-C5)alkylene-O-tosyl and —(CO-
C5)alkylene-O-SO2-(C0-C5(alkyl). More preferably, a reactive group RG is selected from the group consisting of -I, -Br, -CI, -O-tosyl and -O-SO2-(CO-C5(alkyl). 37
Even more preferably, a reactive group RG is selected from the group consisting of -L, -Br, -CI, -O-tosyl and -O-mesyl.
In such a case, a target molecule 1s recovered as it was used before the tagging. For example (the target molecule is in the examples a biomolecule):
NO, NO, biomolecule
Br HS Ss + Sa: ——--
O biomolecule O + HBr
HN HN
Neg Neg light irradiation
NO 9
J * HS: biomolecule
HN
Ne or 38
NO, 25 NO,
SS g-Plomolecule
O TL + Ss biomolecule — > O 206 + 287
HN ° HN HO
N 52° N “0 Oo TL “0
Tosyl as RG light irradiation
NO 9
J + PS piomolecule
HN
N.
O or 39
NO, NO, biomolecule
Br HN , N + FN. oO biomolecule oO + HBr
HN HN
N. 5 N. Ö if NH2 is the nucleophile
Example: N-terminal of a protein light irradiation
NO 9
J + HaN- biomolecule
HN
No
Non-limiting examples for potential corresponding reactive groups on a target molecule are nucleophiles, such as -SH, -OH, -NHz and -NH(C1-C5)alkyl. -S-SO2-(C1-CS)alkyl; -S-S-pyridine
In another preferred embodiment, a reactive group RG is selected from the group consisting of -((CO-
C5)alkylen)-S-SO2-(C1-C5)alkyl and -((CO-C5)alkylen)-S-S-pyridine, ortho meta para no substituents required wherein the pyridine ring may be optionally substituted with one, two, three or four substituents independently from each other selected from the group consisting of (C1-C2)alkyl.
More preferably, a reactive group RG is selected from the group consisting of -S-SO2-(CHz) and -S-S- pyridine wherein the pyridine ring may be optionally substituted with one, two, three or four substituents independently from each other selected from the group consisting of (C1-C2)alkyl.
Even more preferably, a reactive group RG is selected from the group consisting of -S-SO2-(CHs) and -
S-S-pyridine. 40
In such a case, a target molecule 1s recovered as it was used before the tagging. For example (the target molecule is in the examples a biomolecule):
NO,
NO O AS... 2 S§” “biomolecule O 6-0 120
Ss’ > Q + ST + HS. - HO
O biomolecule HN
HN :
NG
No light irradiation
NO 9
HS. biomolecule LL biomolecule ~~ S, + O
SH
HN
No
The corresponding reactive group on a target molecule is -SH. -((CO0-CS)alkylene)-OH.
In another preferred embodiment, a reactive group RG is -((C0-C5)alkylene)-OH.
More preferably, a reactive group RG is -OH.
In such a case, a target molecule is recovered as it was used before the tagging. For example (the target molecule is in the examples a biomolecule): 41
NO, 0
NO, oo biomolecule oO O
OH x © + HO” biomolecule HN
HN :
No
Ne light irradiation
NO 0
O
© + HO” biomolecule
HN
Ne
Non-limiting examples for potential corresponding reactive groups on a target molecule are carboxylic acids. -((C0-CS)alkylen)-NH; and -((C0-CS)alkylen)-NH(C1-CS)alkyl.
In another preferred embodiment, a reactive group RG is selected from the group consisting of -((CO-
C5)alkylen)-NHz and -((CO-C5)alkylen)-NH(C1-C5)alkyl.
More preferably, a reactive group RG is selected from the group consisting of -NHz and -NH(C1-
C2)alkyl.
In such a case, a target molecule is recovered but slightly modified, nevertheless, the tag can be easily removed from the target molecule. For example (the target molecule 1s in the examples a biomolecule): 42
Ku
NO, N biomolecule 0 o H
NH, À
O + HO” “biomolecule ~ HN
HN N.. “0
Nu:
O light irradiation
NO 9
O
© + HN biomolecule
HN
N
O or 43
NO, 0
NO, TW
O
NH,
JO + OCNpiomolecule —— HN
HN isocyanates . y N. Ö
NS light irradiation
NO 9
O
O + Ho” Pomalocues
HN
No
Non-limiting examples for potential corresponding reactive groups on a target molecule are carboxylic acids and isocyanates.
Click chemistry
In another preferred embodiment, a reactive group RG is selected from the group consisting of a terminal azide function selected from the group consisting of -(C0-C5)alkylene-Ns, -O-(C1-C5)alkylene-N3, -
NH-(C1-C5)alkylene-N3, -O-C(O)-(C1-C5)alkylene-N3, and -NH-C(O)-(C1-C5)alkylene-N3; and a terminal alkinyl function selected from the group consisting of -O-(C1-C5)alkylene-ethinyl; -(CO-
C5)alkylene-ethinyl; -NH-(C1-C5)alkylene-ethinyl; -O-C(O)-(CO-C5)alkylene-ethinyl; and -NH-C(O)- (CO-C5)alkylene-ethinyl).
More preferably, a reactive group RG is selected from the group consisting of -(CO-C2)alkylene-N3, -O- (C1-C2)alkylene-N3, -NH-(C1-C2)alkylene-N3, -O-C(O)-(C1-C2)alkylene-N3, and -C(O)-(CI1-
C2)alkylene-N3; and a terminal alkinyl function selected from the group consisting of -O-(Cl- 44
C2)alkylene-ethinyl; | -(CO-C2)alkylene-ethinyl; | -NH-(C1-C2)alkylene-ethinyl; -O-C(O)-(CO-
C2)alkylene- ethinyl; and -NH-C(O)-(CO-C2)alkylene-ethinyl).
In a more preferred embodiment, a reactive group RG is -(CO-C2)alkylene-N3, even more preferred - methylene-N3.
In another more preferred embodiment, a reactive group RG 1s -NH-C(O)-methylene-ethinyl.
In such a case, a target molecule is recovered but slightly modified, nevertheless, the tag can be easily removed from the target molecule. For example (the target molecule is in the examples a modified biomolecule):
NO» O NO» O
N
AN Cy
TH S + N3<piomolecule 7 ON | N
HN HN biomolecule
NG NG light irradiation
NO O
0
O + HN an
HN N biomolecule
No or
NO, NO; 0 O
N NS A
O LA biomolecule O N=N biomolecule + =
HN HN
N. Ô N. Ô light irradiation
NO Ÿ
J M SL
O N
1
N=N biomolecule
HN
N. 104
Non-limiting examples for potential corresponding reactive groups on a target molecule are azides or alkinyls, respectively.
Michael acceptors
In another preferred embodiment, a reactive group RG 1s selected from the group consisting of Michael acceptors such as imide (maleimide), acrylamides and acrylates.
More preferably, a reactive group RG is an imide, even more preferably maleimide.
In such a case, a target molecule is recovered but slightly modified, nevertheless, the tag can be easily removed from the target molecule. For example (the target molecule is in the examples a modified biomolecule): 46
NO» O NO» O
N biomolecule
N Ss
O / + HS biomolecule > 0
O O
HN HN
. No:
Ne O light irradiation
NO 9 o biomolecule o + HN S
HN ©
No or 47
NO, O NO, y biomolecule
Y s
O + MS biomolecule oo AM
HN HN
N. 5 N. 5 light irradiation
NO 9
J y biomolecule + S
O y ; JT
Y=NHorO
No
Non-limiting examples for potential corresponding reactive groups on a target molecule are nucleophiles such as -SH, -OH, -NHz and -NH(C1-C5)alkyl.
PG
Formula (II)
In another preferred embodiment, PG is a photocleavable group of formula (IT)
R2
R4 R1—=
R5 NO,
R3 48
(IT) wherein -* indicates the bond to RG and R1 to RS are as defined hereinabove.
R1
In yet another preferred embodiment, R1 in formula (II) is -(C0-C1)alkylene-C(O)-*, -(CO-
C1)alkylene-C(O)O-(CO-CS)alkylene-*, -(C0-C1)alkylene-OC(O)-(C0-C2)alkylene-*, or -(C1-C2)- alkylene-*.
In yet another preferred embodiment, R1 in formula (II) is -C(O)-*, -C(O)O-(CO-CS5)alkylene-*, -(C1-
C2)alkylene-OC(O)-(CO-C2)alkylene-*, or -CHz-*.
R2 R3 R4 RS
In yet another preferred embodiment, R2 and R3 in formula (IT) independently from each other represent -H, or -(C1-C2)-alkyl.
In yet another preferred embodiment, R2 in formula (II) represents a direct bond to PT, -C(O)NH-*, -O- # -(C0-C5)alkylene-ethinylene-(CO-C5)alkylene-*. {C1-C5)alkylene-", -N((CO-C5)alkyl)H-C(O)-(C0-
C5)alkyl*, -C(0)0O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl-* or
A N
Qi # wherein * indicates the bond between PG and PT and R3 to R5 in formula (II) represent -H, -(C1-C5)- alkyl, -(CO-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -(CO-C5)alkylene-
N((CO-CS5)alky1)((CO-C5)alkyl (-(CO-C5)alkylene-N((CO-C5)alky1)((CO-CS)alky1 1s preferably -
N((CO-CS)alkyl)((CO-C5)alkyl) more preferably R3, R4 and R5 represent independently from each other -H or —(C1-C2)alkyl, even more preferably R3, R4 and RS represent each -H. 49
In one preferred embodiment, wherein R2 represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VIT). In another preferred embodiment, wherein R2 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein
R2 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (IX). In another preferred embodiment, wherein R2 represent a direct bond to PT or encompasses a bond to PT, PT 1s
PT of formula (IX). In another preferred embodiment, wherein R2 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (X).
In yet another preferred embodiment, R3 in formula (IT) represents a direct bond to PT, -C(O)NH-*, -O- * -(C0-C5)alkylene-ethinylene-(C0-C5)alkylene-*. {C1-C5)alkylene-*, -N((CO-CS)alkyl)H-C(O)-(CO-
C5)alkyl*, -C(O)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl-* or
A N
Ci # wherein * indicates the bond between PG and PT and R2, R4 and RS in formula (II) represent -H, -(C1-
C5)-alkyl, -(CO-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -(CO-C5)alkylene-
N((CO-CS)alkyl)((CO-C5)alky1 (-(CO-CS)alkylene-N((CO-CS)alkyl)((CO-CS)alky1 is preferably -
N((CO-CS)alkyl)((CO-C5)alkyl) more preferably R2, R4 and R5 represent independently from each other -H or —(C1-C2)alkyl, even more preferably R2, R4 and RS represent each -H.
In one preferred embodiment, wherein R3 represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VIT). In another preferred embodiment, wherein R3 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein
R3 represent a direct bond to PT or encompasses a bond to PT, PT 1s PT of formula (IX). In another preferred embodiment, wherein R3 represent a direct bond to PT or encompasses a bond to PT, PT is
PT of formula (IX). In another preferred embodiment, wherein R3 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (X).
In yet another preferred embodiment, R4 in formula (II) represents a direct bond to PT, -C(O)NH-*, -O- *, -(CO-C5)alkylene-ethinylene-(CO-C5)alkylene-". {C1-C5)alkylene-", -N((CO-C5)alkyl)H-C(O)-(C0-
C5)alkyl*, -C(O)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl-* or
A N
Qi # wherein * indicates the bond between PG and PT and R2, R3 and R5 in formula (II) represent -H, - (C1-C5)-alkyl, -(CO-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -(CO-
C5)alkylene-N((CO-CS)alkyl)((CO-CS)alky1 (-(CO-CS)alkylene-N((CO-CS)alkyl)((CO-CS)alkyl is preferably -N((CO-C5)alkyl)((CO-C5)alkyl) more preferably R2, R3 and RS represent independently from each other -H or —-(C1-C2)alkyl, even more preferably R2, R3 and R5 represent each -H.
In one preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VII). In another preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein
R4 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (IX). In another preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT, PT is
PT of formula (IX). In another preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (X).
In yet another preferred embodiment, RS in formula (IT) represents a direct bond to PT, -C(O)NH-*, -O- # -(C0-C5)alkylene-ethinylene-(CO-C5)alkylene-*. {C1-C5)alkylene-", -N((CO-C5)alkyl)H-C(O)-(C0-
C5)alkyl*, -C(0)0O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl-* or 51
A N
Qi # wherein * indicates the bond between PG and PT and R2, R3 and R4 in formula (II) represent independently from each other -H, -(C1-C5)-alkyl, -(CO-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH, -
C(O)O-(C1-C5)-alkyl, or -(CO-CS)alkylene-N((CO-CS)alky1)((CO-CS)alkyl (-(CO-CS)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl is preferably -N((CO-C5)alkyl)((CO-CS)alkyl), more preferably R2, R3 and R4 represent independently from each other -H or —(C1-C2)alkyl, even more preferably R2, R3 and R4 represent each -H.
In one preferred embodiment, wherein RS represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VII). In another preferred embodiment, wherein RS represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein
RS represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (IX). In another preferred embodiment, wherein RS represent a direct bond to PT or encompasses a bond to PT, PT is
PT of formula (IX). In another preferred embodiment, wherein R% represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (X).
In yet another preferred embodiment, RS in formula (Il) represents -I, -CI, -Br or -N((CO-
C5)alkyl)((CO-C5)alkyl).
In yet another preferred embodiment, RS in formula (II) represents -N((CO-CS)alky1)((CO-C5)alkyl), preferably -N(ethyl)».
In yet another preferred embodiment, RS in formula (IT) represents -Br.
In yet another preferred embodiment, RS in formula (IT) represents -CI. 52
In yet another preferred embodiment, RS in formula (IT) represents -CI or -Br; and R4 represents a direct bond to PT or encompasses a bond to PT.
In yet another preferred embodiment, RS in formula (II) represents -CI or -Br; and R4 represents a direct bond to PT; and R2 and R3 represent each H.
In yet another embodiment, R4 and R5 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, R4 and RS are direct bonds to PT(-*) (preferably a PT of formula (V) or (VI)).
In yet another embodiment, R4 and R5 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, R4 and RS are direct bonds to PT(-*) (preferably a PT of formula (V) or (VD); and R2 and R3 independently from each other represent -H, or -(C1-C5)-alkyl.
In yet another embodiment, R2 and R4 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, R2 and R4 are direct bonds to PT(-*) (preferably a PT of formula (V) or (VI)).
In yet another embodiment, R2 and R4 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, R2 and R4 are direct bonds to PT(-*) (preferably a PT of formula (V) or (VD); and R3 and R5 independently from each other represent -H, or -(C1-C5)-alkyl.
In yet another embodiment, R3 and R5 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, R3 and RS are direct bonds to PT(-*) (preferably a PT of formula (V) or (VI)).
In yet another embodiment, R3 and RS each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, R3 and RS are 53 direct bonds to PT(-*) (preferably a PT of formula (V) or (VD); and R2 and R4 independently from each other represent -H, or -(C1-C5)-alkyl.
Formula (IIT)
Another preferred embodiment, PG is a photocleavable group of formula (III) *
R7
R4 R9
SN
RS O R6
R8 (II) wherein -* indicates the bond to RG; R4 to R9 are as defined hereinabove.
In one preferred embodiment, R6 represents O.
In another preferred embodiment, R9 represents (C1-C5)alkyl or H, more preferably H.
In yet another preferred embodiment, R4 or RS in formula (III) represent a substituent selected from the group consisting of -Br, -I, -Cl, and -(CO-C5)alkylene-N((CO-C5)alky1)((CO-CS)alkyl).
In yet another preferred embodiment, R4 or RS in formula (III) represent a substituent selected from the group consisting of -Br, -Cl, and -N((C0-C5)alkyl)((C0-C5)alkyl), preferably -N(ethy1)2.
In yet another preferred embodiment, R4 in formula (III) represents a direct bond to PT, -C(O)NH-*, -
O-*, -(C0-C5)alkylene-ethinylene-(CO-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-C5)alkyl)H-C(O)- (C0-C5)alkyl”, -C(0)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, (C0-C5)alkylene-N(C1-C5)alkyl- "or 54
A N
Qi # wherein * indicates the bond between PG and PT and R7, R8 and R5 in formula (III) represent independently from each other -H, -(C1-C5)-alkyl, -(CO-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH, -
C(O)O-(C1-C5)-alkyl, or -(CO-C5)alkylene-N((CO-CS)alkyl)((CO-C5)alky1) (-(CO-CS)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl is preferably -N((CO-C5)alkyl)((CO-C5)alky1), more preferably R7, R8 and R5 represent independently from each other H or (C1-C2)alkyl, even more preferably R7, R8 and R5 represent each -H.
In yet another preferred embodiment, R4 in formula (IIT) represents -N((CO-CS5)alkyl)((CO-CS)alkyl), preferably -N(ethyl)».
In yet another preferred embodiment, R4 in formula (III) represents -N((CO-C5)alkyl)((CO-CS5)alkyl), preferably -N(ethyl)2, and RS represents -H.
In yet another preferred embodiment, R4 in formula (III) represents -N((CO-C5)alkyl)((CO-CS5)alkyl), preferably -N(ethyl)2 and two of R5, R7 and R8 represent -H and the third represents a direct bond to
PT, -C(O)NH-*, -O-", -(C0-C5)alkylene-ethinylene-(C0-C5)alkylene-, -(C1-C5)alkylene-*, -N((CO-
C5)alkyl)H)-C(0)-(C0-C5)alkylen-*, -C(0)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(CO-
C5)alkylene-N(C1-C5)alkyl-" or
A N
Cy #
wherein * indicates the bond between PG and PT.
In yet another preferred embodiment, R4 in formula (III) represents -N((CO-C5)alkyl)((CO-CS5)alkyl), preferably -N(ethyl)2 and two of R5, R7 and R8 represent -H and the third represents a direct bond to
PT, -C(O)NH-*, -O-*, -(C0-C5)alkylene-ethinylene-(C0-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-
C5)alkyl)H)-C(0)-(C0-C5)alkylen-*, -C(0)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(CO-
C5)alkylene-N(C1-C5)alkyl-" or
A N
Qi # wherein * indicates the bond between PG and PT; and RO represents -H.
In one preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VII). In another preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein
R4 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (IX). In another preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT, PT is
PT of formula (IX). In another preferred embodiment, wherein R4 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (X).
In yet another preferred embodiment, RS in formula (IIT) represents -N((CO-CS5)alkyl)((CO-CS)alkyl), preferably -N(ethyl)».
In yet another preferred embodiment, RS in formula (III) represents -N((CO-C5)alkyl)((CO-CS5)alkyl), preferably -N(ethyl)2, and R4 represents -H. 56
In yet another preferred embodiment, RS in formula (III) represents -N((CO-C5)alkyl)((CO-CS5)alkyl), preferably -N(ethyl)2, and two of R4, R7 and R8 represent -H and the third represents direct bond to
PT, -C(O)NH-*, -O-*, -(C0-C5)alkylene-ethinylene-(C0-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-
C5)alkyl)H)-C(0)-(C0-C5)alkylen-*, -C(0)O-*, -OC(O)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(CO-
C5)alkylene-N(C1-C5)alkyl-" or
A N
Qi # wherein ” indicates the bond between PG and PT.
In yet another preferred embodiment, RS in formula (III) represents -N((CO-C5)alkyl)((CO-CS5)alkyl), preferably -N(ethyl)2, and two of R4, R7 and R8 represent -H and the third represents direct bond to
PT, -C(O)NH-*, -O-*, -(C0-C5)alkylene-ethinylene-(C0-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-
C5)alkyl)H)-C(0)-(C0-C5)alkylen-*, -C(0)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(CO-
C5)alkylene-N(C1-C5)alkyl-" or
A N er # wherein * indicates the bond between PG and PT.
In yet another preferred embodiment, RS in formula (IIT) -N((CO-C5)alky1)((CO-C5)alkyl), preferably -
N(ethyl)z, and two of R4, R7 and R8 represent -H and the third represents direct bond to PT, -C(O)NH- # -0-*, -(CO-C5)alkylene-ethinylene-(CO-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-C5)alky1l)H)- 57
C(O)-(CO-C5)alkylen-", -C(0)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(CO-C5)alkylene-N(C1-
C5)alkyl-* or
A N
Cy # wherein * indicates the bond between PG and PT; and RO represents H.
In yet another preferred embodiment, R5 in formula (III) represents a direct bond to PT, -C(O)NH-*, -
O-, -(C0-C5)alkylene-ethinylene-(C0-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-C5)alkyl)H-C(O)- (CO-C5)alky1*, -C(0)0-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, (C0-C5)alkylene-N(C1-C5)alkyl- "or
A N
Qi # wherein * indicates the bond between PG and PT and R7, R8 and R4 in formula (III) represent independently from each other -H, -(C1-C5)-alkyl, -(CO-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH, -
C(O)O-(C1-C5)-alkyl, or -(CO-CS)alkylene-N((CO-CS)alky1)((CO-CS)alkyl) (-(CO-C5)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl is preferably -N((CO-C5)alkyl)((CO-CS)alkyl), more preferably R7, R8 and R4 represent independently from each other -H or -(C1-C2)alkyl, even more preferably R7, R8 and R4 represent each -H.
In one preferred embodiment, wherein RS represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VII). In another preferred embodiment, wherein RS represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein 58
RS represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (IX). In another preferred embodiment, wherein RS represent a direct bond to PT or encompasses a bond to PT, PT is
PT of formula (IX). In another preferred embodiment, wherein RS represent a direct bond to PT or encompasses a bond to PT, PT 1s PT of formula (X).
In yet another preferred embodiment, R7 in formula (III) represents a direct bond to PT, -C(O)NH-*, -
O-*, -(C0-C5)alkylene-ethinylene-(CO-C5)alkylene-*. -(C1-C5)alkylene-*, -N((CO-C5)alkyl)H-C(O)- (C0-C5)alkyl”, -C(0)O-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, (C0-C5)alkylene-N(C1-C5)alkyl- "or
A N er # wherein * indicates the bond between PG and PT and R5, R8 and R4 in formula (III) represent independently from each other -H, -(C1-C5)-alkyl, -(CO-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH,
C(O)O-(C1-C5)-alkyl, or -(CO-C5)alkylene-N((C0-C5)alkyl)((C0-CS)alkyl (-(CO-CS)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl is preferably -N((CO-C5)alkyl)((CO-CS)alkyl), more preferably RS, R8 and R4 represent independently from each other -H or -(C1-C2)alkyl, even more preferably RS, R8 and R4 represent each H.
In one preferred embodiment, wherein R7 represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VII). In another preferred embodiment, wherein R7 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein
R7 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (IX). In another preferred embodiment, wherein R7 represent a direct bond to PT or encompasses a bond to PT, PT is
PT of formula (IX). In another preferred embodiment, wherein R7 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (X). 59
In yet another preferred embodiment, R8 in formula (III) represents a direct bond to PT, -C(O)NH-*, -
O-*, -(C0-C5)alkylene-ethinylene-(CO-C5)alkylene-*, -(C1-C5)alkylene-*, -N((CO-C5)alkyl)H-C(O)- (CO-CS5)alkyl", -C(O)O-*, -OC(O)-*, -C(O)NH-CH2-CH2-NHC(0)-*, (C0-C5)alkylene-N(C1-C5)alkyl- "or
A N
Cy # wherein * indicates the bond between PG and PT and R5, R7 and R4 in formula (III) represent independently from each other -H, -(C1-C5)-alkyl, -(C0-C5)alkylene-O-(C1-C5)-alkyl, -C(O)OH, -
C(O)O-(C1-C5)-alkyl, or -(CO-C5)alkylene-N((C0-C5)alkyl)((C0-CS)alkyl (-(CO-CS)alkylene-N((CO-
C5)alkyl)((CO-C5)alkyl is preferably -N((CO-C5)alkyl)((CO-CS)alkyl), more preferably RS, R7 and R4 represent independently from each other -H or -(C1-C2)alkyl, even more preferably RS, R7 and R4 represent each H.
In one preferred embodiment, wherein R8 represent a direct bond to PT or encompasses a bond to PT,
PT 1s PT of formula (VII). In another preferred embodiment, wherein R8 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (VIII). In another preferred embodiment, wherein
R8 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (IX). In another preferred embodiment, wherein R8 represent a direct bond to PT or encompasses a bond to PT, PT is
PT of formula (IX). In another preferred embodiment, wherein R8 represent a direct bond to PT or encompasses a bond to PT, PT is PT of formula (X).
In yet another preferred embodiment, R4 and RS each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably,
R4 and RS are direct bonds to PT(-") (preferably a PT of formula (V) or (VD).
In yet another preferred embodiment, R4 and RS each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, 60
R4 and RS are direct bonds to PT(-") (preferably a PT of formula (V) or (VI)); and R7 and R8 independently from each other represent -H, or -(C1-C5)-alkyl.
In yet another preferred embodiment, R4 and RS each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably,
R4 and RS are direct bonds to PT(-") (preferably a PT of formula (V) or (VI)); and R7 and R8 independently from each other represent -H.
In yet another preferred embodiment, R7 and R4 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably,
R7 and R4 are direct bonds to PT(-”) (preferably a PT of formula (V) or (VD).
In yet another preferred embodiment, R7 and R4 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably,
R7 and R4 are direct bonds to PT(-") (preferably a PT of formula (V) or (VI)); and R8 and R5 independently from each other represent -H, or -(C1-C5)-alkyl.
In yet another preferred embodiment, R7 and R4 each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably,
R7 and R4 are direct bonds to PT(-") (preferably a PT of formula (V) or (VI)); and R5 and R8 independently from each other represent -H.
In yet another preferred embodiment, R8 and RS each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably,
R8 and RS are direct bonds to PT(-*) (preferably a PT of formula (V) or (VI)).
In yet another preferred embodiment, R8 and RS each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably,
R8 and RS are direct bonds to PT(-*) (preferably a PT of formula (V) or (VI)); and R7 and R4 independently from each other represent -H, or -(C1-C5)-alkyl.
In yet another preferred embodiment, R8 and RS each independently form a direct bond to PT (preferably the PT is PT of formula (V) or (VI)) or comprise a direct bond to said PT. More preferably, 61
R8 and R5 are direct bonds to PT(-*) (preferably a PT of formula (V) or (VI)); and R7 and R4 independently from each other represent -H.
Formula (IV)
In another preferred embodiment, PG 1s a photocleavable group of formula (IV) *
R14 R12
R15 =
NV R13
R16 O R6
R17 (IV) wherein -* indicates the bond to RG and R6 and R12 to R17 are as defined hereinabove.
In another preferred embodiment, R6 represents O and R12 represents S.
In another preferred embodiment, R12 represents O and R6 represents S.
In another preferred embodiment, R12 and R6 each represent S.
In one more preferred embodiment, R6 and R12 each represent O.
In another preferred embodiment, R14, R15, R16 and R17 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-CS)-alkyl, -C(O)OH, -C(0)O-(C1-C5)-alkyl, or -N((CO-CS)alky1)((CO-
CS)alkyl).
In another preferred embodiment, R14, R15 and R17 represent each -H and R16 represents -(C1-C5)- alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -N((C0-C5)alkyl)((CO-C5)alkyl), more preferably -N((CO-C5)alky1)((CO-CS)alkyl). 62
In another preferred embodiment, R13 is a direct bond to PT (-*), -O-*, -(C0-C5)alkylene-ethinylene- (CO-C5)alkylene-", -(C1-C5)alkylene-*, -(C0-C5)alkylene-N(CO-C5)alkyl-* or
A N
Cy # wherein * indicates the bond between PG and PT, more preferably R13 is a direct bond to PT, -O-*, -
NH-*, -(C0-C2)alkylene-ethinylene-(C0-C2)alkylene-*, or -(C1-C2)alkylene-".
In another preferred embodiment, PG is a photocleavable group of formula (IV), wherein R6 and R12 each represent O, R14, R15 and R17 represent each -H and R16 represents -(C1-C5)-alkyl, -O-(C1-
C5)-alkyl, C(O)OH, -C(O)O-(C1-C5)-alkyl, or -N((C0-C5)alkyl)((C0-C5)alkyl), more preferably -
N((CO0-C5)alkyl)((CO-C5)alkyl); and R 13 is a direct bond to PT, -O-*, -NH-* -(CO-C2)alkylene- ethinylene-(C0-C2)alkylene-*, or -(C1-C2)alkylene-*, wherein * indicates the bond between PG and PT; and PT is of formula (VII).
In another preferred embodiment, PG is a photocleavable group of formula (IV), wherein R6 and R12 each represent O, R14, R15 and R17 represent each -H and R16 represents -(C1-C5)-alkyl, -O-(C1-
C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -N((CO-CS)alkyl)((CO-CS)alkyl), more preferably -
N((CO-C5)alky1)((C0-C5)alkyl); and R 13 is a direct bond to PT, -O-*, -NH-*, -(C0-C2)alkylene- ethinylene-(C0-C2)alkylene-*, or -(C1-C2)alkylene-", wherein * indicates the bond between PG and PT; and PT is of formula (VIII).
In another preferred embodiment, PG is a photocleavable group of formula (IV), wherein R6 and R12 each represent O, R14, R15 and R17 represent each -H and R16 represents -(C1-C5)-alkyl, -O-(C1-
C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -N((CO-CS)alkyl)((CO-CS)alkyl), more preferably -
N((CO-C5)alky1)((C0-C5)alkyl); and R 13 is a direct bond to PT, -O-*, -NH-*, -(C0-C2)alkylene- ethinylene-(C0-C2)alkylene-*, or -(C1-C2)alkylene-*, wherein * indicates the bond between PG and PT; and PT is of formula (IX). 63
In another preferred embodiment, PG 1s a photocleavable group of formula (IV), wherein R6 and R12 each represent O, R14, R15 and R17 represent each H and R16 represents -(C1-C5)-alkyl, -O-(C1-C5)- alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -N((C0-C5)alkyl)((C0-C5)alkyl), more preferably -N((CO-
C5)alkyl)((CO-C5)alkyl); and R 13 is a direct bond to PT, -O-*, -NH-*, -(C0-C2)alkylene-ethinylene- (CO-C2)alkylene-*, or -(C1-C2)alkylene-", wherein * indicates the bond between PG and PT; and PT is of formula (IX).
In another preferred embodiment, PG 1s a photocleavable group of formula (IV), wherein R6 and R12 each represent O, R14, R15 and R17 represent each H and R16 represents -(C1-C5)-alkyl, -O-(C1-C5)- alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, or -N((C0-C5)alkyl)((C0-C5)alkyl), more preferably -N((CO-
C5)alkyl)((CO-C5)alkyl); and R 13 is a direct bond to PT, -O-*, -NH-*, -(C0-C2)alkylene-ethinylene- (CO-C2)alkylene-*, or -(C1-C2)alkylene-", wherein * indicates the bond between PG and PT; and PT is of formula (X).
PT
In another preferred embodiment, PT is selected from the group consisting of a compound of formula (V) and (VI).
In another preferred embodiment, PT is selected from the group consisting of a compound of formula (VID), (VII), (IX), IX’) and (X).
In another preferred embodiment, PT is of formula (V)
Rg
Rh Rf
Ri AK +
N + 1 „07
Ra Rb (V) 64 wherein “+” indicates the positions of R4 and RS, or R2 and R4, or R3 and RS, respectively, in formula (ID), preferably the positions of R4 and RS; and Ra, Rb, Rg, Rf, Ri and Rh are as defined hereinabove for formula (V).
In another preferred embodiment, PT is of formula (V) wherein “+” indicates the positions of R4 and
RS, or R7 and R4, or R8 and RS, respectively in formula (III), preferably the positions of R4 and RS; and Ra, Rb, Rg, Rf, Ri and Rh are as defined hereinabove for formula (V).
In another preferred embodiment, Rf and Rg independently from each other represent -C(O)OH; -
C(O)NHz; -OH; -NHz; -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; -(C1-C2)alkyl, -(C5-
C6)cycloalkyl or -H; more preferably Rf and Rg represent -CHs or -H; even more preferably -H.
In another preferred embodiment, Ra, Rb, Rh and Ri independently from each other represent -(C1-
C5)alkyl; more preferably, each of Ra, Rb, Ri and Rh represent -CHs.
In another preferred embodiment, Ra and Rb form together with the C atom to which they are attached a (CS-C6)spiro ring, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH (preferably CH2-OH); and Rh and Ri form together with the C atom to which they are attached a (C5-C6)spiro ring, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH (preferably —CHz-OH).
In another preferred embodiment, PT is of formula (VI) 65
Rk
Rj ++
N + “XX .0
Ra Rb (VD wherein “+” indicates the positions of R4 and RS, or R2 and R4, or R3 and RS, respectively, in formula (ID), preferably the positions of R4 and RS; and Ra, Rb, Rj and Rk are as defined hereinabove for formula (VI).
In another preferred embodiment, PT is of formula (VI) wherein “+” indicates the positions of R4 and
RS, or R7 and R4, or R8 and RS, respectively in formula (III), preferably the positions of R4 and RS; and Ra, Rb, Rj and Rk are as defined hereinabove for formula (VI).
In another preferred embodiment, Ra, Rb, Rj and Rk independently from each other represent -CHz or -
H, more preferably, each of Ra, Rb, Rj and Rk represent -CHa.
In another preferred embodiment, Ra and Rb form together with the C atom to which they are attached a (CS-C6)spiro ring, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH (preferably —CH2-OH); and Rj and Rk form together with the C atom to which they are attached a (C5-C6)spiro ring, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-CS5)alkylene-
OH (preferably —CHz-OH).
In another preferred embodiment, PT is of formula (VII) 66
Reims
Eh |. HE
Rip” NR
PN
Ma Rb (VID) wherein + indicates the position of any one of R2 to RS of formula (II), preferably of R4 or RS of formula (IT) and Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh and Ri are as defined hereinabove for formula (VII).
In another preferred embodiment, PT is of formula (VII) wherein + indicates the position of any one of
R4 to R8 of formula (IIT), preferably of R4 or RS of formula (III) and Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh and Ri are as defined hereinabove for formula (VII).
In another preferred embodiment Ra, Rb, Rh and Ri independently from each other represent -(C1-
CS)alkyl.
In another preferred embodiment Ra and Rh each represent phenyl and Rb and Ri each represent -(C1-
CS)alkyl.
In another preferred embodiment Ra, Rb, Rh and Ri each represent phenyl.
In another preferred embodiment Ra and Rh each represent -(C5-C6)cycloalkyl and Rb and Ri each represent -(C1-C5)alkyl.
In another preferred embodiment Ra, Rb, Rh and Ri each represent -(C5-C6)cycloalkyl.
In another preferred embodiment Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably CH2-OH); and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl 67 and (C1-C5)alkylene-OH (preferably CH2-OH); and Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably CH2-OH); and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably CH2-OH). In one more preferred embodiment,
Ra and Rb as well as Ri and Rh form a CS spiro ring system, optionally substituted with one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-
C5)alkylene-OH (preferably CH2-OH). In another more preferred embodiment, Ra and Rb as well as Ri and Rh form a C6 spiro ring system, optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-
OH (preferably CH2-OH).
In another preferred embodiment, Rc, Rd, and Rg independently from each other represent -H.
In yet another preferred embodiment, Re represents -NHz, -COOH, or -H.
In yet another embodiment, Re represents -NHz, -COOH, or -H and Rf represents -H.
In yet another embodiment, Re and Rf each represent -H.
In yet another embodiment, Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to.
In another preferred embodiment, PT is of formula (VIII)
Rf +
R Re
Rh Rb
Ri | Ra
O
(VII) 68 wherein + indicates the position of any one of R2 to RS of formula (II), preferably of R4 or RS of formula (IT) and Ra, Rb, Re, Rf, Rg, Rh and Ri are as defined hereinabove for formula (VIII).
In another preferred embodiment, PT is of formula (VII) wherein + indicates the position of any one of
R4 to R8 of formula (III), preferably of R4 or RS of formula (IIT) and Ra, Rb, Re, Rf, Rg, Rh and Ri are as defined hereinabove for formula (VIII).
In another preferred embodiment, Ra, Rb, Rh and Ri each represent -(C1-C5)alkyl, preferably -methyl.
In a more preferred embodiment, Ra, Rb, Rh and Ri each represent -(C1-C5)alkyl, preferably -methyl, and Rf and Re represent -H and Rg represents -C(O)OH; -C(O)NHz, -OH, -NHz, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; more preferably Rg represents -H.
In yet another more preferred embodiment, Ra, Rb, Rh and Ri each represent -(C1-C5)alkyl, preferably -methyl, Re represents -NHz, -COOH, or -H and Rf and Rg each represent -H.
In yet another preferred embodiment, Re represents -NHz, -COOH, or -H; and Rf represents -H; or Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to.
In another more preferred embodiment, Ra, Rb, Rh and Ri each represent -(C1-C5)alkyl, preferably - methyl, and Rg represents -H and Re and Rf form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to.
In another preferred embodiment Ra and Rh each represent -phenyl and Rb and Ri each represent -(C1-
CS)alkyl.
In another preferred embodiment Ra, Rb, Rh and Ri each represent -phenyl.
In another preferred embodiment Ra and Rh each represent -(C5-C6)cycloalkyl and Rb and Ri each represent -(C1-C5)alkyl.
In another preferred embodiment Ra, Rb, Rh and Ri each represent -(C5-C6)cycloalkyl. 69
In another preferred embodiment Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably CH2-OH); and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably —CHz-OH); and Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five- membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably CH2-OH); and in case of a six- membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably CHz-OH). In one more preferred embodiment, Ra and Rb as well as Ri and Rh form a C5 spiro ring system, optionally substituted with one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably CH2-OH). In another more preferred embodiment,
Ra and Rb as well as Ri and Rh form a C6 spiro ring system, optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH (preferably —CHz-OH).
In another preferred embodiment, Rg represents -C(O)OH; -C(O)NHz, -OH, -NHz, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-CS)alkyl.
In another preferred embodiment, PT is of formula (IX)
O
HO ye H ry +
Me A
N O
04 À
O OH
70
(IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT),
Mn(II), Yb(III), Fe(II), Co(II), Dy(IlI), Ce(IIl), Tm(IID); and wherein + indicates the position of any one of R2 to RS of formula (ID).
In another preferred embodiment, PT is of formula (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT), Mn(II), Yb(III), Fe(Il), Co(II), Dy (III),
Ce(II), Tm(III); and wherein + indicates the position of any one of R4 to R8 of formula (III).
In another preferred embodiment, PT is of formula (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT), Mn(II), Yb(III), Fe(II), Co(II), Dy(I1I),
Ce(II), Tm(III); and wherein + indicates the position of R13 of formula (IV).
In another preferred embodiment, PT is of formula (IX)
O
HO Da
NW
O
Me /
N O oH
O OH
(IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT),
Mn(II), Yb(III), Fe(II), Co(II), Dy(IlI), Ce(IIl), Tm(III); and wherein + indicates the position of any one of R2 to RS of formula (II).
In another preferred embodiment, PT is of formula (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT), Mn(II), Yb(III), Fe(II), Co(II), Dy(IIM),
Ce(II), Tm(IID); and wherein + indicates the position of any one of R4 to R8 of formula (III). 71
In another preferred embodiment, PT is of formula (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT), Mn(II), Yb(III), Fe(Il), Co(II), Dy (III),
Ce(III), Tm(IID); and wherein + indicates the position of R13 of formula (IV).
In another preferred embodiment, PT is of formula (X)
RI +
NSN Z/
N N
C Me ) „N N°
Rm” \_/ Rn (X) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT),
Mn(II), Yb(III), Fe(II), Co(II), Dy(III), Ce(III), Tm(I1I); and wherein + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; and
RI, Rm and Rn are independently from each other selected from the group consisting of -H, -(C1-
CS)alkyl, -(C1-C5)alkylene-C(O)OH (preferably -CH2-C(O)OH), -(C1-C5)alkylene-C(O)N((CO-
CS)alkyl)z (preferably -CHz-C(O)N((CO-CS)alkyl)2), -(C1-C5)alkylene-N((CO0-C5)alkyl)z (preferably -
CHz-N((CO-CS)alky1)2); and -(C1-C5)alkylene-S-SO2-Me); and + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively.
In one preferred embodiment, Rl, Rm and Rn are independently from each other selected from the group consisting of -H, -(C1-C5)alkyl, -(C1-C5)alkylene-C(O)OH, -(C1-C5)alkylene-S-SO2-(C1-
C5)alkyl, and -(C1-C5)alkylene-C(O)NH-(C1-C5)alkylene-S-SO2-(C1-C5)alkyl. 72
In a more preferred embodiment, Rl, Rm and Rn are independently from each other selected from the group consisting of -H, -CHs, -CH20H, -(C1-C5)alkylene-S-SO2-CHz, —(C1-C2)alkylene-C(O)NH- (C1-C2)alkylene-S-SO2-(C1-C2)alky1.
In an even more preferred embodiment, Rl, Rm and Rn are independently from each other selected from the group consisting of -H, -CHs, -CH20H, -(C1-C2)alkylene-S-SO2-CH;z, -(C1-C2)alkylene-
C(O)NH-(C1-C2)alkylene-S-SO2-CH:z.
In one preferred embodiment, a paramagnetic compound of formula (T) 1s selected from the group consisting of example 1 to 9: 73 ou
N | RT RG
NO
NO» N NO, 2 on RG RG So
Rf Rf > example 1 NL. example 2 9 example 3 0,
N
DZ
HO ON va, .RG ar X HO (ee y I
NV N N
. NV N
ON A A— WS L 0 Xo N 0” So 0 ” À, example 4 OH + example 5 OH example 6 „RG
R1
NGS
O
.RG
R1
CT ON "re
Lx ye "we 9
Me ' LEA, 0 example 8 OH example 7
O example 9
RG can be as defined herein above in the detailed description.
R1 can be as defined herein above in the detailed description. 74
Me can be as defined herein above in the detailed description.
Conjugates
The invention also refers to a conjugate comprising a target molecule, preferably a bio-molecule, which in its state before reacted with a compound according to the invention (i.e. of formula (I) or a precursor of formula (I)) comprises at least one function selected from the group consisting of -SH, -OH, -NHz and -NH(C1-C5)alkyl, -COOH, -N=C=0, -N3, and -ethinyl; and which group can be reacted with a RG of a compound according to the invention. In case the target molecule is a biomolecule, the biomolecule may comprise such a function in its natural state or may be modified to comprise a function selected from the group consisting of -SH, -OH, -NHz and -NH(C1-C5)alkyl, -COOH, -N=C=0, -N3, and -ethinyl before reacting said molecule with a compound according to the invention; e.g. a target molecule may be a protein or a modified protein.
In one preferred embodiment, a target molecule comprises a -SH function in its original state (the term original state refers to the structure of a target molecule before reacting it with a compound according to the invention, e.g. a protein or a modified protein, to which at least one PT-PG can be attached via a sulfide bridge.
In a preferred embodiment, such target molecule conjugates have preferably, depending on the RG and the complementary function of the target molecule, a structure of compound ((XI) :
R2
R4 R1-x — target molecule
R5 NO,
R3 (XI) wherein R1-R5 are as defined herein; and
X represents -S-¥, -O-$, -NH-*, -S-S-% -O-C(O) -*, -NH-C(0)-N((C0-C5)alkyl) -¥, -NH-C(O) -, 75
O
0 ~ N
TA AA 2
N° 8 „N N
N LA. 7 S § , | ‚or © ; wherein -* indicates the bond to a carbon atom of the target molecule (TM).
Preferably, wherein R1 represents -CHz-. Also preferably, wherein R2, R3 and R4 each represent H. Also preferably, wherein RS represents a direct bond to PT, -C(O)NH-*, -O-*, -(C0-C5)alkylene-ethinylene- (CO-C5)alkylene-". <C1-C5)alkylene-", -NH-C(O)-, -C(0)0-*, -OC(O)-", -C(O)NH-CH:-CHa-
NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl- or
A,
Qi # wherein ” indicates the bond between PG and PT. Also preferably, wherein PT is a PT of formula (VII) or (VIII),
More preferably, wherein R1 represents -CHz-, R2, R3 and R4 each represent H, RS represents a direct bond to PT, -C(O)NH-*, -O-*, -(C0-C5)alkylene-ethinylene-(CO-C5)alkylene-*. —(C1-C5)alkylene-*, -
NH-C(0)-*, -C(0)0-*, -OC(0)-*, -C(O)NH-CH2-CH2-NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl-*, or
A,
Cy # wherein * indicates the bond between PG and PT, and wherein PT is a PT of formula (VII) or (VIII). 76
In another preferred embodiment, such target molecule conjugates have preferably, depending on the RG and the complementary function of the target molecule, a structure of compound ((XII) :
TM
X
R7
R4 R9
SNS
R5 O R6
R8 (XII) wherein R4-R9 are as defined herein; and
X represents -S-¥, -O-$, -NH-*, -S-S-% -O-C(O) -*, -NH-C(0)-N((C0-C5)alkyl) -¥, -NH-C(O) -, 0 0 ~ N
Ar N + \ \ 0
N° $ N N
LA. % 0
S > , Or . wherein -* indicates the bond to a carbon atom of the target molecule (TM).
In another preferred embodiment, such target molecule conjugates have preferably, depending on the RG and the complementary function of the target molecule, a structure of compound ((XII) : 77
TM x
R14 R12
R15 >
N R13
R16 O R6
R17 (IV) wherein R6 and R12-R17 are as defined herein; and
X represents -S-$, -0-3, -NH-*, -S-S-3, -O-C(O) -%, -NH-C(O)-N((CO-C5)alkyl) -3, -NH-C(O) -, 0 0 ~ N
Mr N 25 \ 0
N° § „N N
I J A
S > , Or . wherein -* indicates the bond to a carbon atom of the target molecule (TM).
The skilled person will understand that X is the result of a RG of the compound of formula (I) or a RG of the precursor of formula (1)? and a function comprised by the target molecule TM, e.g. a reactive function selected from the group consisting of -SH, -OH, -NHz and -NH(C1-C5)alkyl, -COOH, -N=C=0, -N3, and -ethinyl.
The conjugates can be prepared using a compound of formula (I) or formula (I)’, respectively and are, thus, also subject of the present invention.
In one preferred embodiment, a target molecule is a molecule selected from the group consisting of a protein, a modified protein to comprise a function selected from the group consisting of -SH, -OH, -
NH: and -NH(C1-C5)alkyl, -COOH, -N=C=0, -N3, and -ethinyl, a DNA, a modified DNA to comprise a function selected from the group consisting of -SH, -OH, -NHz and -NH(C1-C5)alkyl, -COOH, -
N=C=0, -Na, and -ethinyl, a RNA, a modified RNA to comprise a function selected from the group consisting of -SH, -OH, -NHz and -NH(C1-C5)alkyl, -COOH, -N=C=0, -N3, and -ethinyl,a polysaccharide, and a modified polysaccharide to comprise a function selected from the group 78 consisting of -SH, -OH, -NHz and -NH(C1-C5)alkyl, -COOH, -N=C=0, -N3, and -ethinyl. The target molecules can be present in their natural state or can be modified before reacting any of them with a compound according to the invention.
In one preferred embodiment, a target molecule is a molecule selected from the group consisting of proteins, DNA, RNA and polysaccharides or a modified versions of any of the foregoing which comprises (or is modified in that it comprises) a -SH group.
In another preferred embodiment, a target molecule is a molecule selected from the group consisting of proteins, DNA, RNA and polysaccharides or a modified versions of any of the foregoing which comprises (or is modified in that it comprises) a -OH group.
In another preferred embodiment, a target molecule is a molecule selected from the group consisting of proteins, DNA, RNA and polysaccharides or a modified versions of any of the foregoing which comprises (or 1s modified in that it comprises) a -COOH group.
Preferred conjugates are those of examples 10 to 32 79
>. & 5 target molecule
RO amy =
SN as
Example 10 Example 11 oe wa Ss target molecule 2 5 . we = ¥ Bog Nar x x N RS 0 y
N
TN og ir
A ( Me 0 an A
Example 12 Ken © Example 13 taraet molscs . = a a wg Ss i .
De Example 15
N t. 8
Example 14 ; 9 target roblecuie x >
Sa £ © NG; ot N «Large molacule
Np Nee
H 3 33 ea - to 16 xarmpie 17
Example 16 fonmengite À 80
AD NO: i CSN 8 | target molecule
N
$ target molecule we. pa,
LX | °
N
3 a
Example 18 NN
HO, __.Q 8 Try ces sn NA § © 3 Sr $ Example 23
Example 19 P
Q <>
N fae! nciscule Æ 4
Rit „oe Hl 8 .
Q 8 target molecule es x N > gg or Ss 73 a Ng
A Example 20 Example 22 81
0" (target raciecule } 2
En nn gy ; a
Fm Y DA © a
Example 23 Example 26 target molecule target mitecuis
REE 9) ô
SX "SCE
OR 2 5 ; Q 3 A
N i oct : - . u
Example 24 J or UR
Example 27
NO, 8 target molecule a N target molacule y . rr xy HN aN 3 So ° Se
Example 25 Example 28 82
7 . N 5-8 Ÿ T # Fr A N NET x NF ON oR
D 5 / 5
Example 28 Example 31 target molecule « N
HR & X
J
Ss) 8 . 2 2
We LÉ i & x 8 “Ny s ya J
Example 30 Example 32
Precursor
The invention also refers to a precursor of a compound of formula (I), wherein PT is replaced by a precursor group PT” which is of formula (IX), (IX) or (X), respectively, but in which the Me is not present (yet).
Thus, one preferred embodiment refers to a precursor of formula (I)’
PT’-PG-RG @
Methods 83
Depending on the functionalization of the chromophore, the photochemical and photophysical properties can be fine-tuned and the wavelength of the absorbed light can be controlled. Accordingly, if two or more
PTs are attached to a target molecule via different PGs that absorb light of different wavelengths site- specific manners, the individual tags could be released selectively and successively, by applying the corresponding wavelength. This procedure would offer the opportunity of studying the effect of multiple paramagnetic tags using only one sample: the tags would be released from the molecule one by one, using light of different wavelength, thus allowing to investigate the effect of each individual tag. In the end, the molecule could be recovered in its native form and used for other experiments. This procedure is particularly promising to study protein complexes, where different tags can be introduced at cysteine located on different molecules before complex assembly. In the following description, the process of releasing only one tag in the presence of a second tag is named orthogonality. Figure 1 describes (a) a target molecule with two photocaged functionalities, (b) the selective release/uncaging of functionality
A by applying light at a wave length, wherein the photocleavable group of A breaks down (Aa), (c) the selective release/uncaging of functionality B by applying light at a wave length, wherein the photocleavable group of B breaks down (As), (d) the release of both functionalities from a target molecule, wherein the PGs break down at the two different wave lengths.
It should be noticed that the photocleavable linker contains a reactive group (RG) at a position in the chemical-structure that permits the tagged molecule to be “un-tagged” by light irradiation affording the studied molecule free from the spin-label. Furthermore, depending on the reaction chosen for the attachment of the paramagnetic tag (for instance a Sn2 between a halide and a free thiol group) the tagged sample can be recovered in its native form after light irradiation (see Scheme 1). 84 target molecule \-SH + paramagnetic / photocleavable RG I => g tag group reaction target molecule }-g photocleavable/” paramagnetic light > target molecule )-SH group tag
Scheme 1: Attachment of a compound of formula (I) to a target molecule via a SN2 reaction and removal of the PT via breakdown of the PG, wherein the released target molecule is unchanged (i.e. has the same functionality (here the -SH group) as before the attachment of a compound of formula (I) according to the invention).
On the other hand, if the tag contains a different RG (reactive group), such for instance azides for click chemistry or Michael acceptors for alkylation, the resulted reaction between the tag and the studied- molecule would not result in exactly the same molecule that was used prior to the attachment of the tag (see Scheme 2).
Click target molecule Y= + paramagnetic" photocleavable }-N, — tag group reaction target molecule 4 N
N° . / _ light op, target molecule —¢ N
N-N photocleavable( paramagnetic H group tag
Scheme 2: Attachment of a compound of formula (I) to a target molecule via a click reaction and removal of the PT via breakdown of the PG, wherein the released target molecule is slightly changed (i.e. the functional group with which the PT was attached (via the PG) to the target molecule is altered after the breakdown of the PG (here the -ethinyl group of the target molecule results in a 85
N
AN
N
: group), but the PT is successfully removed.
In some cases, it is desired to have the molecule derivative in a different form than the native-form due to stability reasons (for instance in the case of resulting in free thiol groups that could further dimerize or react in the presence of oxygen). Therefore, the compounds of formula (I) can be designed to leave a desirable motif as for example showed in Scheme 3.
O
> Sn2 target molecule -sy + ( Paramagnetic(” photocleavable }-g RG — tag group reaction target molecule }S O O ; light / { photocleavable( paramagnetic \ — y. ( target molecule >S OH group tag
Scheme 3: General structure of novel compounds of formula (I) that would recover the studied molecule on an alkylated form after the breakdown of the PG.
In this case, the tagged target molecule would result in an alkylated form of the tagged molecule containing a carboxylic acid at the end of the chain (target molecule-S-CH2-COOH).
Therefore, one aspect of the present invention refers to a method to tag a target molecule with a paramagnetic tag by reacting a target molecule with a molecule of formula (I).
Another aspect refers to a method to analyze a target molecule comprising the steps of 1) reacting the target molecule with at least one molecule of formula (I) resulting in a conjugate; or reacting the target molecule with at least one precursor molecule of formula (1)’ and then adding a Me as defined herein to prepare the PT in the conjugate; 86
2) performing at least one measurement selected from the group consisting of a Paramagnetic
Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement, an electron spin resonance (ESR) measurement, and a fluorescence resonance energy transfer microscopy measurement with the conjugate; 3) removing the paramagnetic tag by exposing the conjugate to light of a wave length which is suitable to break down the PG in the conjugate, preferably exposing the conjugate to light of a wave length between 200 nm and 620 nm, more preferably between 220 nm and 600 nm; 4) optionally recovering the target molecule in its native state (native state = structure of the target molecule before it was reacted with at least one molecule of formula (I)); or recovering the target molecule in an altered form in that the reactive group of the target molecule with which the RG of a molecule of formula (I) reacted, is changed after the breakdown of a PG.
Another aspect refers to a method to analyze a target molecule comprising the steps of 1) reacting the target molecule with at least two different molecules of formula (I), wherein the at least two different molecules differ in that the PG of each molecule breaks down at a different wavelength/in a different wavelength range, resulting in a conjugate with at least two different
PG groups; or reacting the target molecule with at least two different molecules of formula (I) or formula (XI), respectively, wherein the at least two different molecules differ in that the PG of each molecule breaks down at a different wavelength/in a different wavelength range (each within the range between 200 nm and 620 nm, preferably between 220 nm and 600 nm), resulting in a conjugate with at least two different PG groups; if at least one compound of formula (XI) is present, add a Me as defined herein to prepare the PT in the conjugate; 2) performing a Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement with the conjugate with at least two different PG groups; 3) removing one paramagnetic tag by exposing the conjugate with at least two different PG groups to light of a wavelength which is suitable to break down one PG group while the other PG group is not affected by said wavelength; - 4) performing a Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement with the conjugate resulting from step 3); 87
- 5) removing a further paramagnetic tag by exposing the conjugate after step 4) to light of a wavelength which is suitable to break down a second PG group which was not affected by the wavelength of step 3); optionally recovering the target molecule in its native state (native state = structure of the target molecule before it was reacted with at least one molecule of formula (I)); or recovering the target molecule in an altered form in that the reactive group of the target molecule with which the RG of a molecule of formula (I) reacted, is changed after the breakdown of at least one of the two PGs.
In one preferred embodiment, a target molecule for a compound according to the invention is a biomolecule selected from the group consisting of peptide, protein, DNA, RNA and polysaccharide or a derivative of any of the foregoing, wherein any of the foregoing comprises at least one functional group selected from the group consisting of -SH, -OH, -NH2 and -NHR, wherein R is a (C1-C5)alkyl, -COOH, isocynate function, azide function and am alkinyl function before reacting the target molecule with a compound of the invention. A target molecule is a molecule to whish a compound according to the invention should be attached via a reaction of a functional group of said target molecule with the RG of a molecule of formula (I) according to the invention.
Kit
Another aspect of the present invention refers to a kit comprising a compound of formula (I) or a precursor of formula (I), wherein PT is replaced by a precursor group PT” which is of formula (IX), (IX)’ or (X), respectively, but in which the Me is not present.
Preferably, the compound of formula (I) or its precursor of formula (D)? are in a separated container.
Optionally, in case the kit comprises a precursor of formula (I)’, the kit may further comprise the Me in form of a salt in a separated container. Preferably, the counter ion in the salt is selected from the group consisting of chloride, sulphate, bromide, hexafluorophosphate, more preferably chloride. 88
The kit may further comprise a manual instructing the user how to react a compound of formula (I) or a precursor of formula (D’ with a target molecule resulting in a conjugate of the invention or precursor conjugate according to the invention as described herein.
Optionally, in case the kit comprises a precursor of formula (I)’ in a separated container, the kit may further comprise Me in form of a salt thereof, preferably, the counter ion in the salt is selected from the group consisting of chloride, sulphate, bromide, hexafluorophosphate, more preferably chloride, in a separated container a manual instructing the user how to prepare a compound of formula (I)’ by combining Me and the precursor of formula (1)? and/or a manual instructing the user how to prepare a conjugate by combining a precursor conjugate and Me.
The kit may further comprise a manual instructing the user how to remove the PT from a conjugate by applying light of a specific wavelength to the PG in the conjugate. The manual may therefore provide the information which wavelength has to be used to remove the tag.
The kit may further comprise in separated containers with at least one chemical required to perform a reaction of a compound of formula (I) with a target molecule.
The kit may further comprise a manual with information which groups of a target molecule can be used to react a compound of formula (I) with said target molecule. 89
General information
Chemicals and solvents were purchased from commercial suppliers and were used without further purification unless otherwise noted. Petrol ether was distilled prior to use. For thin layer chromatography (TLC) aluminium backed silica gel 60 F254 (from Merck) was used. The visualization occurred by UV fluorescence (254 nm) and/or by staining with KMnO4 (30% in H20). Flash column chromatography was performed using the Reveleris® PREP purification system from BUCHI using silica gel (Eco Flex, particle size 40 um irregular) as specified for each protocol. For characterization of the synthesized small molecules, NMR spectra were recorded on a Bruker Ultrashield 400 MHz Avance-I at 25 °C. Chemical shifts ö are given in ppm relative to residual protonated solvent peaks (CHCI>: 6H = 7.26 ppm, 6C = 77.2 ppm; DMSO: 6H = 2.50 ppm; SC = 39.5 ppm; CH:0H: 6H = 3.31 ppm; SC = 49.0 ppm) and coupling constants (J) are given in Hertz. The following descriptions are used for 'H spectra: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or combinations of these acronyms. NMR spectra of the free radicals were recorded in deuterated methanol (MeOH-d4) by quenching it with solid ascorbic acid in the NMR-tube. Electrospray mass spectra were recorded using either a Waters QTOF-
Premier (Waters Aquity Ultra Performance, ESI) or a LCT Premier (Waters) with the samples solubilized in methanol. The ionization modes, the calculated mass and found mass are given. UV-vis absorption spectra were recorded on an Agilent Technoligies Cary Series UV-Vis spectrophotometer using plastic disposable UV-cuvettes (working range from 230 nm, path length 1.0 cm). EPR analysis were conducted at an X-band (ca 9.5 GHz) CW EPR spectrometer (Brucker Elexsys ES00m) with a Brucker super-high
Q resonator ER4122SHQE at 30 K in methanol. The temperature has been kept constant with EPR900 cryostat from Oxford instruments with helium cooling. chromatography was performed on a Waters
Acquity UPLC system, equipped with a binary solvent manager, sample manager and column heater.
Analysis were performed on an Acquity UPLC® Protein BEH C4 (1.7 um, 2.1 x 50 mm, 1 pkg) column kept at 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and in acetonitrile (B).
A gradient elution was performed at 0.6 mL/min by starting with 95% of eluent A and 5% eluent B, then applying a linear gradient to 00% of eluent A and 100% eluent B in 4.15 min., then applying a 90 linear gradient to 95% of eluent A and 05% eluent B in 0.5 min. The total run time, including re- equilibration, was 05 min and the injection volume was 10uL in positive ionization modes, using the partial-loop fill mode. Mass spectrometry was performed on a Waters Synapt G2-S mass instrument (Waters MS Technologies U.K.) equipped with an electrospray ion (ESI) source operated in positive (ESI+) polarity. All mass spectra data were collected in centroid mode using the MS mode of operation.
The constructs with single cysteine mutant of L7Ae protein from Pf were produced using the Q5 site- directed mutagenesis kit (NEB). The L7Ae protein was expressed and purified by immobilized-metal- ion affinity chromatography (IMAC) through a denaturing purification protocol, as described in detail elsewhere (M. Ahmed, A. Marchanka, T. Carlomagno, Angew. Chem. Int. Ed. 2020, 59, 6866-6873.). Briefly,
The L7Ae protein was expressed in E.coli BL21 (DE3) cells in LB media. The cells were harvested and lysed, the centrifuged and filtered lysate was mixed with guanidinium chloride to achieve protein denaturation. The denaturated lysate containing His-tagged L7Ae was applied to a HisTrap HP column (GE Healthcare) and extensively washed; then L7Aa protein was refolded on the column using long- gradient back into guanidinium-free lysis buffer. Following, the L7Ae was eluted with a 0-100 % gradient of elution buffer. The tagged L7Ae protein was incubated with TEV protease overnight at room temperature in lysis buffer. The cleaved protein was isolated by reverse IMAC using a HisTrap HP column and further purified by size-exclusion chromatography (SEC) with a Superdex75 Increase 10/300 column (GE Healthcare).
To prevent the formation of disulphide bonds between the surface-exposed cysteine side-chains, an excess of DTT was added to the protein solution and incubated for at least 1 hour at room temperature prior to the last SEC purification step. DTT was removed from the protein solution during SEC using a
Superdex75 column equilibrated with DTT-free buffer. Site-directed spin-labeling (SDSL) (i.e. coupling with the nitroxide-based paramagnetic tag) was performed after the final SEC purification step. Ten-fold molar excess of spin-label tag A-I (previously solubilized in methanol) were added to the L7 Ae mutants and the reaction mixture was incubated in the dark overnight at room temperature. The unreacted spin- label was removed during another SEC step.
Outline preparation of tag A-I 91
Br NasCO, OH usa 0SiMe,t-Bu 9, —— © — 0
NO, HO NO, imidazol NO,
OH 97% OH 69% OH 4 Jee NH,
LX oO cl OH OSiMezt-Bu © NO, OC © No, x BF © NO,
HN 57% HN 87% HN
Neg: Nog: No
Nal veu o
NO, “CX
Ne. oO
Scheme 4: Synthesis of tag A containing an iodine as reactive group at the benzylic position (tag A-I).
In order to prepare an example of a compound of formula (I) comprising a metal-chelator as PT, DOTA derivatives can be used. The commercially available DO3 A tert-buthyl ester (CAS: 122555-91-3) was used in order to prepare tag B as showed in the scheme below.
L
A oA vv Ya A
O N N $ N
TL J ONG
Cl N—" Ho
Br SOCh H Le) J 0, — O — N
NO, A NO, Et;N, DCM, rt
OH cl 37% “re
Cl
Scheme 5: Synthesis of spin-label tag B-CI containing metal-chelator DOTA group and chloride at the benzylic position. 92
The removal of the PT by light was shown for three compounds and the results are showed below.
Example 1) Spin-label removal from N-(tert-butoxycarbonyl)-L-cysteine methyl ester upon light irradiation (365 nm).
Figure 2 shows the Spin-label removal from N-(tert-butoxycarbonyl)-L-cysteine methyl ester upon light irradiation (365 nm) measured by liquid chromatography: a) standard N-(fert-butoxycarbonyl)-L- cysteine methyl ester; b) time-zero; c) after 10 min light irradiation (365 nm); d) after 30 min light irradiation (365 nm); e) after 60 min light irradiation (365 nm).
A standard solution of N-(tert-butoxycarbonyl)-L-cysteine methyl ester was prepared in MeOH-d4 and analyzed by HPLC (retention time = 0.54 min, figure 09 a)). The time zero (figure 09 b)) showed no presence of the N-(tert-butoxycarbonyl)-L-cysteine methyl ester. After only 10 minutes of irradiation with 365 nm LED the free cysteine derivative could be observed. The concentration of the analyte increase with the time of irradiation.
Example 2) Remove of the spin label from 2-phenylacetic acid with light irradiation:
ID
Oo light °
MeOH-d © NO, —_—t iO + byproduct
HO
NH xy 2-phenylacetic acid .0” 6.0 mg in MeOH-d4 (1.0 mL)
Scheme 6: Release of 2-phenylacetic acid from a conjugate by light induced breakdown of the PT
Figure 3 shows the 1H-NMR spectra in MeOH-d4 of 2-phenylacetic acid attached to spin-label measured upon light irradiation in minutes: a) zero minutes; b) 30 min under 415 nm; ¢) 30 min under 385 nm; d) min under 365 nm.
The time zero (figure 3 a)) showed broad peaks (due to the tag) and no presence of the 2-phenylacetic acid (benzylic peak at circa 3.6 ppm, according to NMR measurements with commercially available the 93
2-phenylacetic acid). After 30 minutes of irradiation with 365 nm LED the respective acid was detected (figure 3 d)). It was also shown that this photoactive spin-labels could be removed by applying light of lower wavelengths: 385 nm (figure 3 c)) and 415 nm (figure 3 b)). According to fig 3, for the same time of 30 min, the photochemical process was more efficiently using 365, 385 and 415 nm, respectively.
Example 3) Paramagnetic tagging of the protein L7ae-N37C/C70S (M. Ahmed, A. Marchanka, T.
Carlomagno, Angew. Chem. Int. Ed. 2020, 59, 6866-6873.) with tag A-I and subsequent cleavage of tag via irradiation.
Protein 5H + tag Ad messe RESETS Tag photo food: photosmart protein} sath: Demi protein 3 ¢ AR À proto tagàd = 0 :
Ke
Scheme 7: Tagging of the protein L7ae-N37C/C70S with spin-labels (expression and tagging were performed according to the reference provided).
Figure 4: 2D 'H-">’N HSQC spectra of L7ae-N37C/C70S. Left: prior to tagging with tag A-I (diamagnetic control spectrum). Middle: after tagging with tag A-I (paramagnetic spectrum). Right: after photo- cleavage of tag A-I from protein via irradiation (diamagnetic spectrum). Peaks showing significant bleaching in the paramagnetic spectrum are indicated in dashed boxes and labelled with the corresponding residue. The contour-levels have been adjusted to account for changes in sample concentration. Photo-cleavage of tag A-I was achieved by irradiation with light at a wavelength of 365 nm for 6 h, although interim spectra indicated that cleavage had completed in 2 h. After irradiation, the cleaved tag was removed from the sample via a short buffer-exchange prior to acquisition of the diamagnetic spectrum. The spectra were recorded at a temperature of 298 K on a Bruker Avance III-HD
NMR spectrometer operating at a 'H frequency of 850 MHz and equipped with a helium-cooled cryogenic inverse HCN probe-head. The NMR samples were prepared in standard 5-mm-diameter 94 sample tubes (volume ~ 540 pL) using uniformly '*C,!*N-labelled L7ae-N37C/C70S at concentrations of 215-370 uM. The buffer-system was 50 mM HEPES (pH 7.5) and 120 mM NaCl. 10 % v/v D20 was added to the samples for the field-frequency-lock.
Measurement of PREs (paramagnetic relaxation enhancements) is a well-established technique for extracting distance information in the context of structural biology of proteins (and also nucleic acids).
The technique relies on the accelerated nuclear spin relaxation that occurs in molecules with a paramagnetic site, the degree of which for any particular nucleus depends strongly on the distance between the nucleus and the unpaired electron. The most obvious manifestation of the accelerated relaxation due to the electronic paramagnetism is the attenuation of the intensities of peaks in the NMR spectrum. Peaks from nuclei closest to the paramagnetic site typically disappear completely and are said to be “bleached”.
The standard experimental approach is to introduce a paramagnetic site into the protein of interest by covalent attachment of a paramagnetic tag (also called spin-label) via the sulthydryl group of a cysteine residue. The most commonly used tags contain a sterically protected nitroxide group as the paramagnetic center. The appropriate NMR spectrum is then recorded (usually a 2D "H-'*N correlation spectrum); this is called the ‘paramagnetic’ spectrum. The paramagnetic center is then “quenched”, usually by reducing the nitroxide group to a hydroxylamine with ascorbic acid; the tag remains attached to the protein, but is no longer paramagnetic. The same type of NMR spectrum is recorded for a second time (with unchanged acquisition parameters); this is called the ‘diamagnetic’ spectrum. Comparison of the paramagnetic and diamagnetic spectra reveals which peaks were bleached by the paramagnetic tag, and assuming the peaks have been assigned to the respective residues in the protein, gives direct information on which residues are closest to the tagged cysteine residue in the three-dimensional structure of the protein. Quantitative analysis proceeds by first calculating the ratios of the peak intensities between the paramagnetic and diamagnetic spectra, Ipara/ldia.
The approach with the photo-cleavable paramagnetic tag A-I is broadly similar; tagging of the protein via the cysteine sulfhydryl and measurement of the paramagnetic spectrum is as described above. The difference lies in how the electronic paramagnetism is then removed; with tag A-I, the covalent attachment of the tag to the protein is broken by photo-cleavage, which involves irradiating the sample 95 for a few hours with light at a wavelength of 365 nm. The result is that protein returns to the same (diamagnetic) state as that prior to the tagging. The diamagnetic spectrum is recorded on the photo- cleaved sample.
Figure 4 shows three 2D !H-!'*N correlation spectra of the protein L7ae-N37C/C70S. The spectrum on the left-hand side is of the protein before attachment of tag A-I. In the middle is the paramagnetic spectrum, recorded after attachment of tag A-I. On the right-hand side is the diamagnetic spectrum, recorded after photo-cleavage of the tag A-I by irradiation. A subset of residues whose peaks exhibit significant bleaching are indicated within dashed boxes; from the left-hand to the middle spectrum, these peaks are bleached due to the presence of the paramagnetic tag A-I, and then from the middle to the right- hand spectrum, they reappear because the paramagnetic tag has been cleaved from the protein via the irradiation process.
In general, these results showed that molecules containing a spin-label motif connected to a photocleavable linker could be used to tag molecules in order to make them paramagnetic. After performing the desired tasks/analysis with the paramagnetic tagged-molecule (for example, PRE-NMR and EPR, among other analysis), the substance could be irradiated with light in order to remove the paramagnetic tag, recovering the molecule in its native form and/or on a derivative-form depending on the reactive group used to perform the tagging. 96
Hye +, ABS, Kids { ye Nef, BION Ho hy RICHE, reflux, Eh ON FCWARE ON
AN $F gy 7 NO, TE A. 16 Lu Sea 2% san
Sins WEBB AR Gans Das
L{a-aligoe- Smethony-3- nito-phançtf-ethanot
PRG y - By Mt, NEE i Se SNR “ NES ECS sa À Si sy A Ry RRR a NL
El . vo § BAS ES § 7
SN Ie KANAREN ARE Ë Le NE met
AS De Ks ARE ge 5 MESA AAA AAA Sag A
SLR we de dont ¥
SE DR ef & SO BOUT ca STR § à Ce id fa § à
Nagy VF va TE Dem se Que > Ag
Scheme 8: Hypothesis on how to synthesize a tag that could absorb light of higher wavelengths (J. Am.
Chem. Soc. 2019, 141, 9079).
The compound 1-(4-Allyloxy-5-methoxy-2-nitro-phenyl)-ethanol in the scheme above is described in the literature. With this compound in hand, the desired derivative could be prepared in further 6 synthetic steps.
Another derivative that could be prepared in order to absorb light of shorter wavelength and that is very rigidity is shown on the scheme below. 97 sé A ; 45
Be = Fo ve Sadia = en, de an a pd
Fes eds: Np Spek 5 TOY et i î FI Canara sas Be 1 T Nu 20005 Ya i A A=
Tan, AEN , 5 pte on nes i, 2 ane, 205 À *
Y
© i ; ¢ ng EP AREER Nv dee * ¥ 2% à
ET wed hs lor Sons ptet Br Sg 0 8 2 Bie Thal” Meh N 4 FIC en] à Hg * DAT SET + a À
Scheme 9: Hypothesis on how to synthesize a rigid tag that could absorb light of shorter wavelengths.
This protocol involves 8 synthetic steps and it could be modified in case the bromine at the benzylic position is not stable. As for the molecule synthesized previously, during hydrolysis the bromide could be exchanged to hydroxyl group. If this would be the case, the similar approach of protection and deprotection with silanes could be used.
Another approach that would be interesting to try in order to achieve (light) orthogonality would be the connection between the spin-label and the photocleavable linker by an alkyne bond. Alkyne bonds can rotate on the axis but they cannot bend. This is particular important for the technic of PRE-NMR. 98
2 8
Nr SOHN NANG, MOLE Ne Nou sme Sy DOM > {dtode- 2-ritro-shenyii ; i -methanot
PEPPEC fn ft, ; Cx
THF SES, } $a ~ : $ ¥ 3 WR ! des FER i= Ae Pu LI, OH
N J. x Ra Sen X \ ae , £ AON . / as wi La SEL SR Ste A pra Ge sm -
AN Yo, / x BER AN RO,
Scheme 10: Hypothesis on how to synthesize a tag that separated the spin-label to the photocleavable linker by an alkyne (RSC Adv. 2019, 9, 13249).
Compound (4-Iodo-2-nitro-phenyl)-methanol showed on the scheme above could be prepared according to the literature. From this step, desired tag could be prepared by a Sonogashira coupling followed by methodologies described herein.
Precursor 4-(hydroxymethyl)-3-nitrobenzoic acid
LS
0
NO,
OH
Chemical Formula: CgH7NO5
Exact Mass: 197.0324
To a solution of 4-(bromomethyl)-3-nitrobenzoic acid (500 mg, 1.92 mmol, 1.0 eq) in acetone (8.0 mL) a solution of Na2COs (1019 mg, 9.61 mmol, 5.0 eq) in water (8.0 mL) was added. The resulting mixture was heated at reflux temperature for 5 h. After allowing the mixture to reach r.t., the acetone was removed in vacuo. The remaining solution was washed with ether (1 x 10 mL), acidified to pH 1 using 1.0 M 99
HCl) solution and extracted with EtOAc (3 x 10mL). The combined organic layers were washed with water (1 x 10 mL), dried over Na:SOs and concentrated in vacuo to yield 4-(hydroxymethyl)-3- nitrobenzoic acid (368 mg, 1.87 mmol, 97%) as a brown solid. The 'H NMR data is in accordance with the previously described in the literature!”
R= 0.32 (DCM/MeOH (9:1) [UV, KMnO4].
IH NMR (400 MHz, DMSO-d6) ô = 13.53 (bs, 1H), 8.48 (d, J=1.7, 1H), 8.27 (dd, J= 8.1, 1.7, 1H), 7.98 (d, J=8.1, 1H), 5.70 (bs, 1H), 4.89 (s, 2H). 4-(((tert-butyldimethylsilyl)oxy)methyl)-3-nitrobenzoic acid 6 o 1 i oO 47 2 NO,
OH
Chemical Formula: C44H,,NO;Si
Exact Mass: 311.1189 (1) Under argon, imidazole (1600 mg, 23.50 mmol, 6.0 eq) was added to a solution of fert-butyl dimethylsilyl chloride (2361 mg, 15.66 mmol, 4.0 eq) in dry DMF (15.0 mL) at 0 °C. The resulting solution, was stirred for 5 minutes and, after that, a solution of 4-(hydroxymethyl)-3-nitrobenzoic acid (772 mg, 3.92 mmol, 1.0 eq) in dry DMF (15.0 mL) was added dropwise. The reaction mixture was allowed to reach r.t. and stirred for 19 h. After that, DMF was removed in vacuo and the remaining crude was suspended in DCM (150.0 mL). The organic layer was washed with saturated solution of NH4Cl(ag) (1 x 30.0 mL), followed by water (1 x 30.0 mL). After that, the organic layer was dried over NazSO4, and the solvent was evaporated in vacuo to afford the intermediate tert-butyldimethylsilyl 4-(((tert- butyldimethylsilyl)oxy)methyl)-3-nitrobenzoate as a brown oil, which was used in the next step without further purification. (11) The crude intermediate was diluted in THF (20.0 mL) followed by the addition of a saturated solution of NaHCO3(aq) (20.0 mL). The reaction was stirred at r.t. for 2 h and after that, THF was evaporated in 100 vacuo. The remaining aqueous solution was acidified to pH 1 using 1.0 M HCl) solution and the acidic aqueous layer was extracted with DCM (1 x 120.0 mL). After that, the aqueous layer was discarded and the organic layer was washed with brine (1 x 60.0 mL), dried over Na2SO4 and the solvent was removed in vacuo to afford 4-(((tert-butyldimethylsilyl)oxy)methyl)-3-nitrobenzoic acid (846 mg, 2.72 mmol, 69%) as brown solid.
R= 0.57 (DCM/MeOH (9:1) [UV, KMnO4].
IH NMR (400 MHz, CDCIs) 5 = 8.82 (d, J=1.7, 1H, Ar-3-H), 8.42 — 8.35 (m, 1H, Ar-5-H), 8.08 (d,
J=8.2, 1H, Ar-6-H), 5.17 (s, 2H, a-H), 1.00 — 0.96 (m, 9H, Si-C-(CH:)3), 0.18 — 0.14 (m, 6H, Si-C- (CH3)2).
BC NMR (100 MHz, CDCI-) § = 169.11 (Ar-CO-NH), 146.61 (Ar-2-C), 144.25 (Ar-5-C), 134.93 (Ar- 1-C), 129.04 (Ar-4-C), 128.78 (Ar-6-C), 126.56 (Ar-3-C), 62.37 (a-C), 26.03 (Si-C-(CH:):), 18.51(Si-
C), -5.26 (Si-C-(CHs)2).
HRMS (ESI) m/z: [C14 H2o N Os Si] calcd.: 310.1111; found: 310.1109
Synthesis of derivative tag A-OSMDBT 101
$ 1 A o A oO
JK
HN, + 4 3 2
A N. sy Co
Chemical Formula: C>3H538N5O;5Si
Exact Mass: 464.2581 (1) Under argon, 4-(((tert-butyldimethylsilyl)oxy)methyl)-3-nitrobenzoic acid (160 mg, 0.51 mmol, 1.0 eq) was dissolved in SOCI2 (2445 mg, 1.50 mL, 20.55 mmol, 40.0 eq), and the solution was stirred under reflux for 1 h. After that, SOCI2 was evaporated in vacuo to afford crude acyl chloride intermediate as dark-brown oil, which was used in the next step without further purification. (11) Under argon, a solution of 4-amino-TEMPO (88 mg, 0.51 mmol, 1.0 eq) in DCM (2.0 mL) was prepared and the temperature of the solution was dropped to 0 °C. After that, TEA (114 mg, 0.16 mL, 20.55 mmol, 1.13 eq) was added and the resulting mixture was stirred at 0 °C for 5 min. After this procedure, a freshly prepared solution of the crude acyl chloride intermediate in DCM (2.0 mL) was added dropwise. The reaction mixture was allowed to reach r.t. and it was further stirred for 4.0 hours.
After that, the temperature of the reaction was dropped to 0 °C and water (4.0 mL) was added. The resulting mixture was transfer to a separatory funnel and the organic layer was separated. The water layer was washed with DCM (2 x 8.0 mL) and the combined organic layers were washed with brine (1 x 8.0 mL), dried over Na2COs and the volatiles were evaporated in vacuo. The residue was purified by flash column chromatography using 12 g silica gel column as stationary phase and a mixture of petrol ether (PE) and ethyl acetate (EtOAc) (Flow 25 mL per minute; mixture gradient: from pure PE to pure EtOAc in 25 minutes) as eluent to afford product (125 mg, 0.27 mmol, 52%) as white solid.
Re 0.73 (PE/EtOAc (1:1) [UV, KMnO4].
IH and *C NMR of the free radical was recorded by quenching it with excess of ascorbic acid:
IH NMR (400 MHz, MeOD) 5 = 8.52 (d, J=1.8, IH, Ar-3-H), 8.15 (dd, J=8.2, 1.8, 1H, Ar-5-H), 7.96 (d,
J=8.2, 1H, Ar-6-H), 5.13 (s, 2H, 0-H), 4.44 — 4.37 (m, 1H, 4 -CH), 1.97 — 1.89 (m, 2H, 3 -CH>), 1.66 (t, 102
J=12.5, 2H, 3 -CH;), 1.27 (d, J=18.0, 12H, 2’ -C-((CH5)2)»), 0.97 (s, 9H, Si-C-(CH:):), 0.15 (s, 6H, Si-
C-(CH5)2).
BC NMR (100 MHz, MeOD) 5 = 167.04 (Ar-CO-NH), 148.22 (Ar-2-C), 141.89 (Ar-1-C). 135.74 (Ar- 4-C), 133.08 (Ar-5-C), 129.60 (Ar-6-C), 124.63 (Ar-3-C), 61.58 (a-C), 50.76 (2'-C), 45.24 (3°-C), 42.93 (4'-C), 32.08 (N-C-(CHz)2), 26.32 (S1-C-(CH:)3), 20.34 (N-C-(CH:)2), 19.21 (Si-C), -5.42 (S1-C-(CHa)2).
HRMS (ESI) m/z: [C23 H3s N3 Os Si + Na] calcd.: 487.2478; found: 487.2478
Synthesis of derivative tag A-OH 6 o 5% 70H
O
DZ No,
HN, A 4] 32
A N. >
Chemical Formula: C47H254N305
Exact Mass: 350.1716
Under argon, derivative tag A-OSMDBT (41 mg, 0.88 mmol, 1.0 eq) was diluted in dried THF (2.0 mL) and stirred at 0 °C for 5 min. After that, a solution of 1 M TBAF in THF (46 mg, 0.18 mmol, 2.0 eq) was added and the mixture was allowed to reach r.t. and stirred for 45 min. After that, brine (1.5 mL) was added to the reaction and the resulting mixtrure was extracted with EtOAc (3 x 15.0 mL). The combined organic layers were dried over Na2SO4 and the solvent was evaporated in vacuo. The residue was purified by flash column chromatography using 12 g silica gel column as stationary phase and a mixture of petrol ether (PE) and ethyl acetate (EtOAc) (Flow 25 mL per minute; mixture gradient: from pure PE to pure
EtOAc in 25 minutes) as eluent to afford product (27 mg, 0.077 mmol, 87%) as slightly-yellow solid.
Re 0.17 (PE/EtOAc (1:1) [UV, KMnO4].
IH and *C NMR of the free radical was recorded by quenching it with excess of ascorbic acid: 103
IH NMR (400 MHz, MeOD) 5 = 8.52 (d, J=1.7, 1H, Ar-3-H), 8.16 (dd, J=8.2, 1.8, 1H, Ar-5-H), 7.98 (d,
J=8.2, 1H, Ar-6-H), 5.00 (s, 2H, a-H), 4.40 (m, 1H, 4 -CH), 1.94 (dd, J=12.9, 3.8, 2H, 3'-CH>), 1.66 (t,
J=12.3, 2H, 3'-CH>), 1.27 (d, J=18.3, 12H, 2'-C-((CH5)2)»).
BC NMR (100 MHz, MeOD) 5 = 167.12 (Ar-CO-NH), 148.42 (Ar-2-C), 142.60 (Ar-1-C), 135.51 (Ar- 4-C), 133.12 (Ar-5-C), 129.78 (Ar-6-C), 124.59 (Ar-3-C), 61.79 (a-C), 49.62 (2'-C), 44.61 (3 -C), 42.56 (4 -C), 31.17 (N-C-(CH:)2), 20.36 (N-C-(CH3)2).
HRMS (ESI) m/z: [C17 H24 N3 Os + Na]* caled.: 373.1614; found: 373.1611
Synthesis of derivative tag A-CI 6 a
J CI
SA
72 NO,
HN, A 41 32 x N
IY 70
Chemical Formula: C47H43CIN304°
Exact Mass: 368.1377
Under argon, derivative tag A-OH (27 mg, 0.077 mmol, 1.0 eq) was diluted in DCM (3.0 mL) and pyridine (27 mg, 28 uL, 0.35 mmol, 4.5 eq) was added. The resulting mixture was stirred at r.t. for 5 min and, after that, the temperature was dropped to 0 °C. After another 5 min stirring at 0 °C, SOCI» (23 mg, 14 uL, 0.19 mmol, 2.5 eq) was added dropwise and the mixture was allowed to reach r.t. and it was stirred for 1 h. Subsequently, the reaction mixture was cooled to 0 °C and quenched with water (5.0 mL) and extracted with DCM (3 x 7.0 mL). The combined organic layers were dried over Na2SOs and the solvent was evaporated in vacuo. The residue was purified by flash column chromatography using 4 g silica gel column as stationary phase and a mixture of petrol ether (PE) and ethyl acetate (EtOAc) (Flow 15 mL per minute; mixture gradient: from pure PE to pure EtOAc in 25 minutes) as eluent to afford product (16 mg, 0.043 mmol, 57%) as red solid/film. 104
Re 0.49 (PE/EtOAc (1:1) [UV, KMnO4].
IH and 3C NMR of the free radical was recorded by quenching it with excess of ascorbic acid:
IH NMR (400 MHz, MeOD) 5 = 8.49 (d, J=1.7, 1H, Ar-3-H), 8.13 (dd, J=8.0, 1.8, 1H, Ar-5-H), 7.83 (d,
J=8.1, 1H, Ar-6-H), 5.04 (s, 2H, a-H), 4.38 — 4.32 (m, 1H, 4 -CH), 1.95 — 1.85 (m, 2H, 3'-CH>), 1.63 (t,
J=12.4, 2H, 3'-CH>), 1.25 (d, J=16.0, 12H, 2°-C-((CH3)2)2).
BC NMR (100 MHz, MeOD) § = 166.51 (Ar-CO-NH), 149.54 (Ar-2-C), 137.35 (Ar-1-C), 136.61 (Ar- 4-C), 133.26 (Ar-5-C), 133.21 (Ar-6-C), 125.16 (Ar-3-C), 49.85 (2 -C), 45.35 (3° -C), 43.09 (4 -C), 43.03 (a-C), 32.30 (N-C-(CH3)2), 20.32 (N-C-(CHs)2).
HRMS (ESI) m/z: [C17 Hzs CI Ns Os + Na]* caled.: 391.1275; found: 391.1277
Synthesis of tag A-1 6 a 1 1 à 72 No,
HN, A 41 32 ; N > 0
Chemical Formula: C47H3IN504°
Exact Mass: 460.0733
Under argon, derivative tag A-Cl (16 mg, 0.043 mmol, 1.0 eq) was solubilized in dry acetone (1.5 mL).
The solution was stirred for 5 min at r.t. and after that Nal (33 mg, 0.22 mmol, 5.0 eq) was added at once.
The resulting mixture was stirred at r.t. for 15 h and after that acetone was evaporated in vacuo. The crude was diluted in DCM (5.0 mL) and the resulting organic solution was extracted with water (5.0 mL).
The organic layers were dried over Na2SO4 and the solvent was evaporated in vacuo to afford photo active spin label (15 mg, 0.033 mmol, 92%) as orange-red solid. 105
Re 0.49 (PE/EtOAc (1:1) [UV, KMnO4].
IH and *C NMR of the free radical was recorded by quenching it with excess of ascorbic acid:
IH NMR (400 MHz, MeOD) 5 = 8.46 (d, J=1.7, 1H, Ar-3-H), 8.04 (dd, J=8.0, 1.8, 1H, Ar-5-H), 7.72 (d,
J=8.1, 1H, Ar-6-H), 4.87 (s, 2H, a-H), 4.41 — 4.35 (m, 1H, 4 -CH), 1.97 — 1.89 (m, 2H, 3'-CH>), 1.66 (t,
J=12.6, 2H, 3’-CH»), 1.27 (d, J=17.2, 12H, 2'-C-((CH>)2)2).
BC NMR (100 MHz, MeOD) 5 = 166.62 (Ar-CO-NH), 148.84 (Ar-2-C), 139.54 (Ar-1-C), 136.60 (Ar- 4-C), 133.59 (Ar-5-C), 133.32 (Ar-6-C), 125.56 (Ar-3-C), 49.57 (2 -C), 45.06 (3 -C), 42.90 (4 -C), 31.87 (N-C-(CHz)2), 20.34 (N-C-(CH:)2), -1.25 (a-C).
HRMS (ESI) m/z: [C17 Hzs I N3 Oa + Na]* calcd.: 483.0631; found: 483.0633
Other spin labels containing various reacrive groups were prepared from compound tag A-I and tag A-
OH.
Derivative tag A-SSO2Me
O oO
HN NO; Na Lo HN NO;
I Ng” ~ s 9
MeOH De ve e
N \ ° 0 6
Under argon, derivative tag A-I (15 mg, 0.033 mmol, 1.0 eq) was solubilized in dry methanol (1.0 mL).
The solution was stirred for 5 min at r.t. and after that methanethiolsulfonate (8.7 mg, 0.065 mmol, 2.0 eq) was added at once. The resulting mixture was stirred at r.t. for 3 h and after that the crude was diluted with brine (2.0 mL) and the resulting solution was extracted with EtOAc (3 x 5.0 mL). The combined organic layers were dried over Na2SO4 and the solvent was evaporated in vacuo to afford product (7 mg, 0.016 mmol, 97%) as red film. 106
Re 0.27 (PE/EtOAc (1:1) [UV, KMnO4].
HRMS (ESI) m/z: [Cis Has N3 Os S2 + Na] calcd.: 467.1161; found: 467.1159
Derivative tag A-N3
O O
EC HN NO,
NaNz
AN Na 0) MeOH-d;
N N
Ô Ô
Chemical Formula: C47H23NgO4
Exact Mass: 375.1781
Under argon, derivative tag A-I (2 mg, 0.004 mmol, 1.0 eq) was solubilized in deuterated methanol (MeOH-d4) (1.0 mL). The solution was stirred for 5 min at r.t. and after that sodium azid (3.8 mg, 0.043 mmol, 10.0 eq) was added at once. The resulting mixture was stirred at r.t. for 10 h and after that the product was observed by 1H-NMR in quantitative yield.
Re 0.48 (PE/EtOAc (1:1) [UV, KMnO4].
IH and 3C NMR of the free radical was recorded by quenching it with excess of ascorbic acid:
IH NMR (400 MHz, MeOD) 5 8.54 (d, J = 1.8 Hz, 1H), 8.16 (dd, J = 8.1, 1.8 Hz, 1H), 7.81 (d, J = 8.1
Hz, 1H), 4.89 (s, 2H), 4.41 — 4.33 (m, 1H), 1.88 (ddd, J = 11.5, 3.9, 1.7 Hz, 2H), 1.63 (t, J = 12.5 Hz, 2H), 1.23 (d, J = 19.1 Hz, 12H).
BC NMR (100 MHz, MeOD) & 166.59, 149.34, 136.94, 135.71, 133.29, 131.91, 125.09, 52.62, 49.62, 45.52, 43.18, 32.52, 20.34. 107
Derivative Tag A-NH2
O oO
NH
MeOH
N N
0 6
Chemical Formula: C47H25N404
Exact Mass: 349.1876
Under argon, derivative tag A-I (2 mg, 0.004 mmol, 1.0 eq) was solubilized in dry methanol (1.0 mL).
The solution was stirred for 5 min at r.t. and after that a solution of ammonia in methanol (7 N) was added (300 uL, 2.1 mmol) at once. The resulting mixture was stirred at r.t. for 10 h and after that the product was observed by 1H-NMR in quantitative yield.
R= 0.03 (EtOAc/Et3N (1:0.05) [UV, KMnO4].
IH NMR of the free radical was recorded by quenching it with excess of ascorbic acid:
IH NMR (400 MHz, MeOD) § 8.70 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.9 Hz, 1H), 4.48 (s, 2H), 4.43-4.35 (m, 1H), 1.90 (d, J = 10.6 Hz, 2H), 1.63 (d, J = 12.3 Hz, 2H), 1.26 (d, J = 16.6 Hz, 12H).
HRMS (ESI) m/z: [C17 Has Na O4+ HJ” calcd.: 350.1952; found: 350.1953
Synthesis of tag A-cystein derivative 108
O
HNO
S ~ O
I Ÿ nf ~
LT KUN à CH3CN 6
Chemical Formula: CogH3gN4OgS
Exact Mass: 567.2489
Under argon, N-(fert-butoxycarbonyl)-L-cysteine methyl ester (1.5 mg, 0.007 mmol, 1.0 eq) was diluted in dry acetonitrile (0.5 mL). After stirring for 5 min, NaH 60% dispersion in mineral oil (0.3 mg, 0.007 mmol, 1.0 eq) was added at room temperature. The resulting solution was stirred at room temperature for 20 min, followed by the addition of a solution of the tag A-I (3 mg, 0.007 mmol, 1.0 eq) in dry acetonitrile (0.5 mL). The resulting mixture was stirred at room temperature for 10 h and the solvent was evaporated in vacuo. The residue was purified by flash column chromatography using 4 g silica gel column as stationary phase and a mixture of petrol ether (PE) and ethyl acetate (EtOAc) (Flow 15 mL per minute; mixture gradient: from pure PE to pure EtOAc in 25 minutes) as eluent to afford product ( mg, 0.043 mmol, 57%) as red solid/film
Re 0.51 (PE/EtOAc (1:1) [UV, KMnO4].
HRMS (ESI) m/z: [C26 H39 N4 Os S + Na]’ calcd.: 590.2386; found: 590.2377
Derivative tag A-O-tosyl 109
O oO
NO NO,
HN 2 TsOTs HN 0
OH — og
I
N N
Ô O
Chemical Formula: C»4Hz0N307S
Exact Mass: 504.1804
Under argon, derivative tag A-OH (2 mg, 0.006 mmol, 1.0 eq) was diluted in a solution of DCM/DMF (1.5 + 1.05mL) and trimethylamine (5.6 mg, 4 uL, 0.029 mmol, 5.0 eq) was added. The resulting mixture was stirred at r.t. for 5 min and, after that, the temperature was dropped to 0 °C. After another 5 min stirring at 0 °C, p-toluenesulfonic anhydride (5.6 mg, 0.017 mmol, 3.0 eq) was added and the mixture was allowed to reach r.t. and it was stirred for 5 h. After that, the product was observed on TLC and by
HRMS (isolated yield not determined).
Re 0.63 (PE/EtOAc (1:1) [UV, KMnO4].
HRMS (ESI) m/z: [C24 H30 N3 O7S + Na]’ calcd.: 527.1702; found: 527.1698
Derivative tag A-Br
O oO
NO, NO,
HN KBr HN
LX CT Lx .
N N
0 o
Chemical Formula: C47H23BrN304
Exact Mass: 412.0872 110
Under argon, derivative tag A-CI (17 mg, 0.046 mmol, 1.0 eq) was solubilized in dry acetone (1.5 mL).
The solution was stirred for 5 min at r.t. and after that KBr (27.4 mg, 0.23 mmol, 5.0 eq) was added at once. The resulting mixture was stirred at r.t. for 15 h and after that acetone was evaporated in vacuo.
The crude was diluted in DCM (5.0 mL) and the resulting organic solution was extracted with water (5.0 mL). The organic layers were dried over NazSO4 and the solvent was evaporated in vacuo to afford product (9 mg, 0.022 mmol, 47%) as a red film.
Re 0.49 (PE/EtOAc (1:1) [UV, KMnO4].
HRMS (ESI) m/z: [C17 H23 N3 O4 Br + Na] calcd.: 435.0770; found: 435.0771
Synthesis of a variation of the spin-label
OH
OSiMe t-Bu LX OSiMezt-Bu
O 1 O
NO, — 9 NO;
NJ
O
Chemical Formula: C»3H37N2O;Si
Exact Mass: 465.2421 (1) Under argon, 4-(((tert-butyldimethylsilyl)oxy)methyl)-3-nitrobenzoic acid (5 mg, 0.016 mmol, 1.0 eq) was dissolved in SOCI2 (821 mg, 0.5 mL, 6.9 mmol, 430.0 eq), and the solution was stirred under reflux for 1 h. After that, SOCI2 was evaporated in vacuo to afford crude acyl chloride intermediate as dark-brown oil, which was used in the next step without further purification. (11) Under argon, a solution of 4-Hydroxy-TEMPO (2.8 mg, 0.016 mmol, 1.0 eq) in DCM (1.0 mL) was prepared and the temperature of the solution was dropped to 0 °C. After that, TEA (8.1 mg, 11.2 uL, 0.08 mmol, 5.0 eq) was added and the resulting mixture was stirred at 0 °C for 5 min. After this procedure, a freshly prepared solution of the crude acyl chloride intermediate in DCM (1.0 mL) was added dropwise. 111
The reaction mixture was allowed to reach rt. and it was further stirred for 4.0 hours. After that, the product was observed on TLC and by HRMS (isolated yield not determined).
Re 0.86 (PE/EtOAc (1:1) [UV, KMnO4].
HRMS (ESI) m/z: [C23 H37 N2 O6 Si + Na] caled.: 488.2319; found: 488.2331
Derivative tag A-OCH2-phenyl
ID
OH cl o 9 NO, OT 9 NO,
OH i : OH i
Chemical Formula: C4gH13NOg
Exact Mass: 315.0743 of
NH,
N o
LO veu o
NO,
Xp” „N 0
Chemical Formula: Co5H3gN3Og
Exact Mass: 468.2135 (1)Under argon, 4-(hydroxymethyl)-3-nitrobenzoic acid (110 mg, 0.588 mmol, 1.0 eq) was dissolved in dry DCM/DMF (2.0 + 1.0 mL) at 0 °C, followed by addition of trimethylamine (178 mg, 246 uL, 1.76 112 mmol, 3.0 eq). The resulting solution, was stirred for 5 minutes at 0 °C and, after that, phenylacetyl chloride (118 mg, 101 pL, 0.764 mmol, 1.3 eq) was added dropwise. The reaction mixture was allowed to reach r.t. and stirred for 24 h. After that, water was added (0.5 mL) and the resulting solution was stirred for 1 h. After that, the organic solvent was removed in vacuo and the remaining crude was suspended in 1.0 mL of acetonitrile/water (1:1). The residue was purified by flash column chromatography using 12 g C18-reversed phase silica gel column as stationary phase and a mixture of water and acetonitrile both containing 0.05% of TFA as eluent (Flow 24 mL per min. Mixture gradient: water (0.05% TFA) / acetonitrile (0.05% TFA)) from 95:5 to 5:95 in 24 minutes) to afford 3-nitro-4-((2- phenylacetoxy)methyl)benzoic acid (93 mg, 0.295 mmol, 50%) as white solid.
R= 0.20 (DCM/MeOH (9:1) [UV, KMnO4].
IH NMR (400 MHz, DMSO) 5 8.52 (d, J = 1.7 Hz, 1H), 8.23 (dd, J=8.1, 1.7 Hz, 1H), 7.74 (d, J = 8.1
Hz, 1H), 7.38 — 7.25 (m, 5H), 5.53 (s, 2H), 3.83 (s, 2H). 13C NMR (100 MHz, DMSO) 8 170.76, 165.26, 147.14, 136.14, 134.06, 133.99, 131.59, 129.45, 129.41, 128.43, 126.97, 125.38, 62.49, 40.06.
HRMS (ESI) m/z: [C16 Hı3 N O6 + Na]’ calcd.: 338.0641; found: 338.0641 (11) Under argon, 3-nitro-4-((2-phenylacetoxy)methyl)benzoic acid (30 mg, 0.095 mmol, 1.0 eq) was dissolved in SOCI (906 mg, 0.55 mL, 7.61 mmol, 80.0 eq), and the solution was stirred under reflux for 1 h. After that, SOCI2 was evaporated in vacuo to afford crude acyl chloride intermediate as dark-brown oil, which was used in the next step without further purification. (111) Under argon, a solution of 4-amino-TEMPO (13 mg, 0.076 mmol, 0.8 eq) in DCM (1.0 mL) was prepared and the temperature of the solution was dropped to 0 °C. After that, TEA (21.2 mg, 29.2 uL, 0.21 mmol, 2.2 eq) was added and the resulting mixture was stirred at 0 °C for 5 min. After this procedure, a freshly prepared solution of the crude acyl chloride intermediate in DCM (1.0 mL) was added dropwise.
The reaction mixture was allowed to reach r.t. and it was further stirred for 5.0 hours. After that, the temperature of the reaction was dropped to 0 °C and water (2.0 mL) was added. The resulting mixture 113 was transfer to a separatory funnel and the organic layer was separated. The water layer was washed with
DCM (2 x 4.0 mL) and the combined organic layers were washed with brine (1 x 8.0 mL), dried over
Na:COs and the volatiles were evaporated in vacuo. The residue was purified by flash column chromatography using 4 g silica gel column as stationary phase and a mixture of petrol ether (PE) and ethyl acetate (EtOAc) (Flow 12 mL per minute; mixture gradient: from pure PE to pure EtOAc in 12 minutes) as eluent to afford product (22 mg, 0.047 mmol, 49%) as red film.
Re 0.44 (PE/EtOAc (1:1) [UV, KMnO4].
IH NMR (400 MHz, CDCls) § 8.60-7.89 (bs, 2H), 7.52 (bs, 1H), 7.36 (bs, 5H), 5.61 (bs, 2H), 3.79 (bs, 2H). 13C NMR (100 MHz, CDCl3) § 170.17, 146.78, 135.32, 132.76, 128.79, 128.27, 126.95, 62.37, 40.85.
HRMS (ESI) m/z: [C2s H30 N3 Os + Na] caled.: 491.2032; found: 491.2037 114
Synthesis of tag B-CI ok oA
X Ya X
Br . Cl N N 0 i + ©
Ke ; 0, Ke YX \ \ Ho
OH Cl —
H
9 ok oA
X CC
0 0 N N (IF n—" [ x”
CI
Chemical Formula: C34Hs4CIN5Og
Exact Mass: 711.3610 (1) Under argon, 4-(bromomethyl)-3-nitrobenzoic acid (25 mg, 0.096 mmol, 1.0 eq) was dissolved in
SOCI2 (800 mg, 0.49 mL, 6.73 mmol, 70.0 eq), and the solution was stirred under reflux for 1.5 h. After that, SOCI2 was evaporated in vacuo to afford crude 4-(chloromethyl)-3-nitrobenzoyl chloride intermediate (in one step) as dark-brown oil, which was used in the next step without further purification. (11) Under argon, a solution of DO3A tert-buthyl ester (CAS: 122555-91-3) (50 mg, 0.096 mmol, 1.0 eq) in DCM (1.0 mL) was prepared and the temperature of the solution was dropped to 0 °C. After that, TEA (41 mg, 56 uL, 0.404 mmol, 4.2 eq) was added and the resulting mixture was stirred at 0 °C for 5 min.
After this procedure, a freshly prepared solution of the crude acyl chloride intermediate in DCM (1.0 115 mL) was added dropwise. The reaction mixture was allowed to reach r.t. and it was further stirred for 4.0 hours. After that, the volatiles were evaporated in vacuo and the residue was purified by flash column chromatography using 12 g silica gel column as stationary phase and a mixture of dichloromethane and methanol (9:1) (Flow 25 mL per minute) as eluent to afford product (25 mg, 0.035 mmol, 37%) as white solid.
R= 0.50 (DCM/MeOH (9:1) [UV, KMnO4].
HRMS (ESI) m/z: [C34 Hs4 Ns Oo C1+ HJ" caled.: 712.3688; found: 712.3682
Synthesis of tag B-1 ok ok oA A
N N
X ( 0 X ( 0 0 X“ N — og N \ Mr 5 \ AN n—" —
O Oo
CI I
Chemical Formula: C34H54IN509
Exact Mass: 803.2966
Under argon, tag B-CI (18 mg, 0.025 mmol, 1.0 eq) was solubilized in dry acetone (1.5 mL). The solution was stirred for 5 min at r.t. and after that Nal (19 mg, 0.127 mmol, 5.0 eq) was added at once. The resulting mixture was stirred at r.t. for 15 h and after that acetone was evaporated in vacuo. The crude was diluted in DCM (5.0 mL) and the resulting organic solution was extracted with water (5.0 mL). The 116 organic layers were dried over NazSO4 and the solvent was evaporated in vacuo to afford product (20 mg, 0.025 mmol, 98%) as orange film.
R= 0.52 (DCM/MeOH (9:1) [UV, KMnO4].
HRMS (ESI) m/z: [C34 Hs4 Ns Oo I+ Na] caled.: 826.2864 ; found: 826.2887
Selected examples of UV-Vis spectroscopy
Figure 5 shows absorption spectra (245-800 nm) and chemical structure of derivative tag A-OSMDBT [Anm (e/M* cm’) + 5% = 365(203)] (a), derivative tag A-OH [Anm (e/M* cm™) + 5% = 365(222)] (b), derivative tag A-Cl [nm (e/M* em!) + 5% = 365(103)] (c), and tag A-I [nm (e/M em!) + 5% = 365(95)] (d). All samples were measured in methanol at 20 °C.
Selected examples of Electron Paramagnetic Resonance (EPR) spectra
Figure 6 shows X-band EPR spectra and chemical structures of derivative tag A-OSMDBT (a), derivative tag A-OH (b), tag A-I (c), and derivative tag A-CI (d). All samples were measured in methanol at room temperature.
Irradiation chamber
Samples were irradiated in the NMR-tube (outer-diameter of 5 mm, made from borosilicate 5.1 glass,
ASTM Class B) using a commercially available irradiator chamber equipped with a cooling fan. Figure 7 shows an ilrradiator chamber obtained from Optobiolabs GmbH.
The light-source irradiates homogeneously 5 cm of the NMR-tube. The experiments were done in a Cold
Room (temperature 6 °C) and the temperature inside the chamber was stabilized at 15 °C during the experiments.
The LEDs used were Kingbright ATS2012UV365 (with a maximum emission of 365 nm), Kingbright
ATS2012UV385 (with a maximum emission of 385 nm) and Kingbright ATS2012UV415 (with a 117 maximum emission of 415 nm). Figure 8 shows the emission spectra of the LEDs used (temperature of measurement 25 °C).
The power density that reaches the NMR-tube was calculated by the supplier to be approximately 60 mW/cm? (milliwatts per square centimetre). 118
Claims (15)
1. A paramagnetic compound for formula (I) PT-PG-RG (I) wherein PT is a chemical system comprising at least one unpaired electron which system is connected with PG via one or two bonds, preferably a system selected from the group consisting of nitroxides, chelating agents; RG is a reactive group selected from the group consisting of -(CO-C5)alkylene-halide (preferably -(CO-C5)alkylene-I, -(CO-C5)alkylene-Br, -(C0-C5)alkylene-Cl); -(CO-C5)alkylene-OTs (i.e. -O- tosyl, 1e- 0O-S02-CsHs-CH3); -(CO-C5)alkylene-O-SO2-(C1-C5)alkyl (preferably -(CO- C5)alkylene-OMs (i.e. -(CO-C5)alkylene-O-mesyl, i.e. -(CO-C5)alkylene-O-SO2z-CHz); -(CO- C5)alkylene-O-C(O)-phenyl; -(CO-C5)alkylene-S-SO2-(C1-C5)alkyl; -(CO-C5)alkylene-OH; - (CO-C5)alkylene-NHz; -(C0-C5)alkylene-NH(C1-C5)alkyl; a terminal alkinyl or azide function,
(e.g., -(CO-C5)alkylene-N3; -O-(C1-C5)alkylene-N3; -NH-(C1-C5)alkylene-N3;-O-C(O)-(CO- C5)alkylene-N3; -N((CO-C5)alkyl)-C(O)-(C1-C5)alkylene-N3 (preferably -NH-C(O)-(C1- C5)alkylene-N3), -O-(C1-C5)alkylene-ethinyl; -(CO-C5)alkylene-ethinyl; N)(CO-CS5)alkyl)-(C1- C5)alkylene-ethinyl (preferably -NH-(C1-C5)alkylene-ethinyl); -O-C(O)-(CO-C5)alkylene- ethinyl; -N((C0-C5)alkyl)-C(O)-(C0-CS5)alkylene-ethinyl | (preferably = -NH-C(O)-(CO- C5)alkylene-ethinyl); Michael acceptor residues such as imidyl, preferably maleimidyl, acrylamidyls, acrylates, and -(C0-C5)alkylene-S-S-pyridinyl (the term encompasses ortho, meta and para positions of the S-bond to the nitrogen of the pyridine ring) wherein the pyridine ring may be optionally substituted independently from each other with one, two, three or four (C1- C5)alkyl; and 119
PG is a photocleavable group selected from the group consisting of A) formula (IT) R2 R4 R1— R5 NO, R3 (In) wherein -* indicates the bond to RG; R1 is either -(CO-C5)alkylene-C(O)-*, -(CO-C5)alkylene-C(O)O-(CO-C5)alkylene-*, -(C1- C5)alkylene-OC(O)-(CO-C5)alkylene-*, -(C1-C5)N(CO-C5)alky1)(C(O)-(CO-C5)alkylene-* and -(C1-C5)O(C(O)-(CO-C5)alkylene-* or -(C1-C5)-alkylene-*, R2, R3, R4 and RS independently from each other represent -H, -(C0-C5)alkylene-halide, (preferably -(CO-CS)alkylene-I, -(CO-C5)alkylene-Br, -(CO-C5)alkylene-CI, more preferably -I
(Le. -(C0)alkylene-I), -Cl or -Br, even more preferably -Br or -CI), -(C1-C5)-alkyl, -(CO- C5)alkylene-O-(C1-C5)-alkyl, -C(O)O(CO-CS)alkyl) (preferably COOH), -C(O)O-(C1-C5)- alkyl, -(C0-C5)alkylene-N((CO0-C5)alkyl)((CO-C5)alkyl) (preferably -N((CO-CS5)alkyl)((CO- C5)alkyl), i.e. -(C0-C5)alkylene is -CO-alkylene), a direct bond to PT (-*), -C(O)NH-*, -O-*, -(CO- C5)alkylene-ethinylene-(CO-C5)alkylene-*. {C1-C5)alkylene-*, -N((C0-C5)alkyl)H-C(O)-(CO- C5)alkyl*, -C(0)0-*, -O-C(O)-, -C(O)NH-CH2-CH2-NHC(O)-*, -(C0-C5)alkylene-N(C1- C5)alkyl-* or 120
A, Qi
# wherein * indicates the bond between PG and PT; provided that either one of R2, R3, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)) or comprises a bond to PT (-*) (preferably a bond to PT of formula (VII), (VII), (IX), (IX), or (X)), preferably, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VID), (IX), (IX), or (X)) or comprises a bond to PT (preferably a bond to PT of formula (VID), (VII), (IX), (IX), or (X)); or R4 and R5 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R4 and RS are direct bonds to PT(-*) (preferably a PT of formula (V) or (VI)) , and R2 and R3 independently from each other represent -H, -(C1- C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO- C5)alkyl)((CO-C5)alkyl), preferably R2 and R3 independently from each other represent -H, - (C1-C5)-alkyl, R2 and R4 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R2 and R4 are direct bonds to PT (-*) (preferably PT of formula (V) or (VI)), and RS and R3 independently from each other represent -H, -(C1- C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO0-CS5)alkylene-N((CO- C5)alkyl)((CO-C5)alkyl), preferably RS and R3 independently from each other represent -H, - (C1-C5)-alkyl,
121
R3 and R5 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (preferably, R3 and R5 are direct bonds to PT (-*) (preferably PT of formula (V) or (VI)) , and R2 and R4 independently from each other represent -H, -(C1- C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO- C5)alkyl)((CO-C5)alkyl), preferably RS and R3 independently from each other represent -H, - (C1-C5)-alkyl, In one preferred embodiment, R1 is selected from the group consisting of -(CO-C5)alkylene- C(O)-*, -(CO-C5)alkylene-C(O)O-(CO-CS)alkylene-*, -(CO-C5)alkylene-OC(O)-(CO- C5)alkylene-*, and -(C1-C5)-alkylene-*, more preferred selected from the group consisting of - C(O)-*, -(CO-C5)alkylene-C(O)O-(CO-CS)alkylene-*, -(CO-C5)alkylene-OC(O)-(CO- C5)alkylene-*, and (C1-C2)-alkylene; or even more preferably R1 is (C1-C2)-alkylene); or B) formula (III) * R7 R4 R9 S R5 O R6 R8 (II) wherein -* indicates the bond to RG; R4 and RS are as defined in A); R6 represents O or S; 122
R7 and R8 independently from each other represent -H, -(CO-C5)alkylene-halide, (preferably - (CO-CS)alkylene-I, -(CO-C5)alkylene-Br, -(CO-C5)alkylene-CI, more preferably -I (Le. (CO)alkylene-I), - Cl or -Br, even more preferably -Br or -CI), -(C1-C5)-alkyl, -(CO-C5)alkylene- O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-CS)alky1)((CO- C5)alkyl), a direct bond to PT (-*), -C(O)NH-, -O-*, -(C0-C5)alkylene-ethinylene-(CO- C5)alkylene-*. {C1-C5)alkylene-*, -N((CO-C5)alkyl)H-C(O)-(CO-CS)alky1l*, -C(0)O-*, -OC(O)- # -C(O)NH-CH2-CH2-NHC(0)-*, -(C0-C5)alkylene-N(C1-C5)alkyl- or A, Qi # wherein * indicates the bond between PG and PT; and RO represents -(C1-C5)alkyl or -H; provided that either one of R4, RS, R7 or R8 is a direct bond to a PT (preferably a PT of formula (VII), (VIII), (IX), IX"), or (X)) or comprises a bond to PT (-) (preferably a PT of formula (VII), (VIII), (IX), (IX), or (X)), preferably, R4 or RS is a direct bond to a PT (preferably a PT of formula (VII), (VIID), (IX), (IX), or (X)) or comprises a bond to PT (preferably a PT of formula (VII), (VIII), (IX), 1X’), or (X)); Preferably, R7 or R8 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)- alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(C0-C5)alkylene-N((C0-C5)alkyl)((CO-C5)alkyl)); or R4 and R5 each independently form a direct bond to PT (preferably of formula (V) or (VI)) or comprise a direct bond to said PT (-*) (preferably, R4 and R5 are direct bonds to PT (preferably of formula (V) or (VI)) , and R7 and R8 independently from each other represent -H, -(C1-C5)- 123 alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO- C5)alky1)((CO-C5)alkyl); or R7 and R4 each independently form a direct bond to PT (-*) (preferably a PT of formula (V) or (VD) or comprise a direct bond to said PT (-*) (preferably, R7 and R4 are direct bonds to PT (preferably of formula (V) or (VI)) , and RS and R8 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO- C5)alky1)((CO-C5)alkyl); or R8 and R5 each independently form a direct bond to PT (-*) (preferably a PT of formula (V) or (VI) or comprise a direct bond to said PT (preferably, RS and R8 are direct bonds to PT (preferably of formula (V) or (VD)) , and R4 and R7 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1-C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO- CS)alky1l)((CO-CS)alky1); or C) formula (IV)
* R14 R12 R15 > NV R13 R16 O R6 R17 (IV) wherein -* indicates the bond to RG; R6 and R12 independently from each other represent O or S; 124
R14, R15, R16 and R17 independently from each other represent -H, -(C1-C5)-alkyl, -O-(C1- C5)-alkyl, -C(O)OH, -C(O)O-(C1-C5)-alkyl, -(CO-C5)alkylene-N((CO-CS)alky1)((CO-CS)alky1); and R13 is a direct bond to PT (-), -O-*, -(CO-C5)alkylene-ethinylene-(C0-C5)alkylene-*. —(C1- C5)alkylene-*, -(C0-C5)alkylene-N(C0-C5)alkyl-* or A, Cy # wherein * indicates the bond between PG and PT.
2. The paramagnetic compound according to claim 1, wherein PT is elected from the group consisting of nitroxides, chelating agents.
3. The paramagnetic compound according to claim 1 or 2, wherein PG is selected from the group consisting of formula (II) or formula (III); and PT is selected from the group consisting of formula (V) Rg Rh Rf RA, N + Pt 07 KX Ra Rb 125
(V) wherein “+” indicates the positions of R4 and RS, or R2 and R4, or R3 and RS, respectively, in formula (II); or the positions of R4 and RS, or R7 and R4, or R8 and RS, respectively in formula (IIT), respectively, and Rf and Rg independently from each other represent -H, -(C1-C5)alkyl, -(C5-C6)cycloalkyl, - C(O)OH, -C(O)NH2, -OH, -NH2, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or -phenyl (preferably, if one of Ra or Rb is a -(C5-C6)cycloalkyl, the other substituent is -(C1-C5)alkyl); or Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (Cl- C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (Cl- C5)alkylene-OH; Ri and Rh independently from each other represent (C1-C5)alkyl, (C5-C6)cycloalkyl or phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1- C5)alkylene-OH; or 126 formula (VI) which is attached to PG via two bonds (see definitions of R2 to RS in formula (ID); and definitions of R4 to R8 in formula (III), respectively): Rk Rj ++ + N
“À .O Ra Rb (VI) wherein “+” indicates the position of R4 and R5, or R2 and R4, or R3 and RS, respectively, in formula (IT); or the positions of R4 and RS, or R7 and R4, or R8 and RS, respectively in formula (II), respectively, and Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1- C5)alkylene-OH; Rj and Rk independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Rj or Rk is a cycloalkyl, the other substituent is (C1-C5)alkyl); or Rj and Rk form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and 127
(C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1 - C5)alkylene-OH; or formula (VIT) which 1s attached to PG via one bond Reine Ry RE Rio” Rea 07 X ke ° Ha R&B (VII) wherein + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (IIT), respectively; and Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1- C5)alkylene-OH; Ri and Rh independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or 128
Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1- C5)alkylene-OH; Rc, Rd, and Rg independently from each other represent -H, -(C1-C5)alkyl, -(CS- C6)cycloalkyl, -C(O)OH, -C(O)NHz, -OH, -NH_, or -pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; and Rf and Re, respectively, either independently from each other represent -(C1-C5)alkyl, -(CO- C5)alkylene-N((CO-C5)alkyl)((CO-CS)alkyl), -(CO-C5)alkylene-COO(CO-CS)alkyl or -H; or Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to; or formula (VIII) Rf + R Re Rh Rb Ri | Ra O (VII) wherein + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; and wherein 129
Ra and Rb independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ra or Rb is a cycloalkyl, the other substituent is (C1-C5)alkyl); or Ra and Rb form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1- C5)alkylene-OH; Ri and Rh independently from each other represent -(C1-C5)alkyl, -(C5-C6)cycloalkyl or - phenyl (preferably, if one of Ri or Rh is a cycloalkyl, the other substituent is (C1-C5)alkyl); or Ri and Rh form together with the C atom to which they are attached a (C5-C6)spiro ring system, optionally substituted with, in case of a five-membered spiro ring system, one, two, three or four substituents independently selected from the group consisting of (C1-C5)alkyl and (C1-C5)alkylene-OH; and in case of a six-membered spiro ring system, one, two, three, four or five substituents independently selected from the group consisting of (C1-C5)alkyl and (C1- C5)alkylene-OH; Rg represents -H, -(C1-C5)alkyl, -(C5-C6)cycloalkyl, -C(O)OH; -C(O)NHz, -OH, -NHz, or - pyridinyl (the term encompasses ortho, meta and para positions of the bond to the nitrogen of the pyridine ring) wherein the pyridinyl ring may be optionally substituted independently from each other with one, two, three or four (C1-C5)alkyl; Rf and Re, independently from each other represent (C1-C5)alkyl or H; or Rf and Re form a bond resulting in a double bond between the two carbon atoms to which Rf and Re are attached to: or formula (IX)
130
© OH HO Da TN O + Me A N O oH O OH (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(III), Mn(II), Yb(III), Fe(II), Co(II), Dy(IID), Ce(III), Tm(IID); and wherein + indicates the position of any one of R2 to RS of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; or formula (IX) O HO T NW O Me N O 04 À O OH (ax) (IX) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(III), Mn(IT), Yb(IIT), Fe(II), Co(II), Dy(IID), Ce(III), Tm(III); and 131 wherein + indicates the position of any one of R2 to R5 of formula (II); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; or formula (X)
RI + NN N N C Me ) „N N Rm \__/ Rn (X) wherein Me represents a chelated metal ion, preferably selected from the group consisting of Gd(IIT), Mn(ID), Yb(IIT), Fe(II), Co(II), Dy (III), Ce(III), Tm(IID); and wherein + indicates the position of any one of R2 to R5 of formula (II); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively; and RI, Rm and Rn are independently from each other selected from the group consisting of -H, - (C1-C5)alkyl, -(C1-CS)alkylene-C(O)O(C0-CS)alkyl, -(C1-CS)alkylene-C(O)N((CO- C5)alkyl)2, -(C1-C5)alkylene-N((CO-C5)alky1)2, (C1-C5)alkylene-S-SO2-(C1-C5)alkyl, and - (C1-C5)alkylene-C(O)NH-(C1-C5)alkylene-S-SO2-(C1-C5)alkyl; and + indicates the position of any one of R2 to R5 of formula (IT); or any one of R4 to R8 of formula (III); or R13 of formula (IV), respectively. 132
4. The paramagnetic compound according to claim 1 or claim 2, wherein PG is of formula (IV); and PT is selected from the group consisting of formula (VII), (VIII), (IX), (IX) or (X) wherein + indicates the position of R13 of formula (IV) and all definitions are those as described in claim
3.
5. The paramagnetic compound according to any one of claims 1 to 3, wherein PG is of formula (ID) R2 R4 R1—= R5 NO, R3 (In wherein R1 to RS and -* are as defined in claim 1.
6. A precursor of a paramagnetic compound according to any one of claims 1 to 5 of formula (I)’ PT’-PG-RG Formula (I)’ wherein PG and RG are as defined in any one of the preceding claims and PT” is of formula (IX), (IX) or (X), respectively, but in which the Me is not present. 133
7. A paramagnetic compound according to any one of claims 1 to 5 or a precursor of formula (I)’ according to claim 6, wherein PT is of formula (VII) or (VIII) and wherein the definitions are those as described in claim 3.
8. A paramagnetic compound according to any one of claims 1 to 5 or 7 or a precursor of formula (D’ according to any one of claims 6 or 7, wherein RG is selected from the group consisting of - I, -Br, -Cl, -O-tosyl and -O-mesyl, -S-SO2-(CHs), -S-S-pyridine, -OH, -NHz, -NH(C1-C2)alkyl, and -maleimideyl.
9. A paramagnetic compound according to any one of claims 1 to 3 or 5 or 8, wherein PG is selected from the group consisting of formula (II); and R1 in formula (IT) is -CHz-.
10. A paramagnetic compound according to any one of claims 1 to 3 or 5 or 7 to 9, wherein PG is selected from the group consisting of formula (IT) and (III); wherein R1 in formula (II) is -CH2-; and PT is selected from the group consisting of formula (VII) or formula (VIII), wherein Ra, Rb, Rh and Ri each represent methyl and Rc to Rg in formula (VII) each represent -H and Re to Rg in formula (VIII) each represent -H, respectively.
11. A conjugate of formula (XI) R2 R4 R1-x— target molecule R5 NO, R3 (XD wherein R1-R5 are as defined ın any of the preceding claims; and 134
X represents -S-¥, -O-S, -NH-*, -S-S-$, -O-C(O) -*, -NH-C(O)-N((CO-C5)alkyl) -¥, -NH-C(O) +, O 0 ~ N Ar X 25 u:
N° $ „N N AS ;
5, ! a wherein -* indicates the bond to a carbon atom of the target molecule (TM); or a conjugate of formula ((XIT)
TM X R7 R4 R9 NS R5 LC R8 (XII) wherein R4-R9 are as defined in any one of the preceding claims; and X represents -S-¥, -O-S, -NH-*, -S-S-$, -O-C(O) -*, -NH-C(O)-N((CO-C5)alkyl) -¥, -NH-C(O) +, O O ~ N a4 N Ar N \ 0
NT 8 „N N ' LA ;
§ , | ‚or © ; wherein -* indicates the bond to a carbon atom of the target molecule (TM); or a conjugate of formula ((XIII)
135
TM x R14 R12 R15 > N R13 R16 O R6 R17 (XIII) wherein R6 and R12-R17 are as defined in any one of the preceding claims; and X represents -S-%, -0-3, -NH-*, -S-S-3, -O-C(O) -%, -NH-C(O)-N((CO-C5)alkyl) =, -NH-C(O) -, 0 0 ~ N Ar N Ar x NT $ „N N N A $ O 5 S > , Or . wherein -% indicates the bond to a carbon atom of the target molecule (TM).
12. A method to analyze a target molecule comprising the steps of 1) reacting the target molecule with at least one molecule of formula (I) resulting in a conjugate; or reacting the target molecule with at least one precursor molecule of formula (I)’ and then adding a Me as defined herein to prepare the PT in the conjugate; 2) performing at least one measurement selected from the group consisting of a Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement, an electron spin resonance (ESR) measurement, and a fluorescence resonance energy transfer microscopy measurement with the conjugate; 3) removing the paramagnetic tag by exposing the conjugate to light of a wave length which is suitable to break down the PG in the conjugate, preferably exposing the conjugate to light of a wave length between 200 nm and 620 nm, more preferably between 220 nm and 600 nm; 136
4) optionally recovering the target molecule in its native state (native state = structure of the target molecule before it was reacted with at least one molecule of formula (I)); or recovering the target molecule in an altered form in that the reactive group of the target molecule with which the RG of a molecule of formula (I) reacted, is changed after the breakdown of a PG.
13. A method to analyze a target molecule comprising the steps of 1) reacting the target molecule with at least two different molecules of formula (I), wherein the at least two different molecules differ in that the PG of each molecule breaks down at a different wavelength/in a different wavelength range, resulting in a conjugate with at least two different PG groups; or reacting the target molecule with at least two different molecules of formula (I) or formula (XI), respectively, wherein the at least two different molecules differ in that the PG of each molecule breaks down at a different wavelength/in a different wavelength range which are both within the range between 200 nm and 620 nm, more preferably between 220 nm and 600 nm, resulting in a conjugate with at least two different PG groups; 1f at least one compound of formula (XI) is present, add a Me as defined herein to prepare the PT in the conjugate; 2) performing a Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement with the conjugate with at least two different PG groups; 3) removing one paramagnetic tag by exposing the conjugate with at least two different PG groups to light of a wavelength which is suitable to break down one PG group while the other PG group is not affected by said wavelength; 4) performing a Paramagnetic Relaxation Enhanced - Nuclear Magnetic Resonance (PRE-NMR) measurement with the conjugate resulting from step 3); 5) removing a further paramagnetic tag by exposing the conjugate after step 4) to light of a wavelength which is suitable to break down a second PG group which was not affected by the wavelength of step 3); optionally recovering the target molecule in its native state (native state = structure of the target molecule before it was reacted with at least one molecule of formula (I)); or recovering the target molecule in an altered form in that the reactive group of the target molecule with which the RG of a molecule of formula (I) reacted, is changed after the breakdown of at least one of the two PGs. 137
14. A kit comprising a compound of formula (I) according to any of claims 1 to 5 or 7 to 10 or a precursor of formula (I)’ according to any one of claims 6 to 8 in a separated container.
15. The kit of claim 14 further comprising a manual instructing the user how to react a compound of formula (I) or a precursor of formula (I)’ with a target molecule resulting in a conjugate or a precursor conjugate. 138
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