Tetrafunctional photoaffinity labels based on Nakanishi's m-nitroalkoxy-substituted phenyltrifluromethyldiazirine

Article abstract of DOI:10.1002/1521-3773(20020703)41:13<2293::AID-ANIE2293>3.0.CO;2-9

Two generally applicable reagents for photoaffinity probes have been developed. They contain an m-nitrophenyl ether function with a trifluoromethyldiazirine side chain (photophore group), as well as a biotin tag for the identification of labeled proteins

Full text of DOI:10.1002/1521-3773(20020703)41:13<2293::AID-ANIE2293>3.0.CO;2-9

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Table 1. Inhibition of p38 MAP kinase, cytokine release, and cytochrome P450 [4] a) G. Mloston, T. Gendek, H. Heimgartner, Helv. Chim. Acta 1998, 81,
isoforms byselected compounds.
1585 1595; b) R. Bartnik, W. Hahn, G. Mloston, Rocz. Chem. 1977, 51,
1747.
[5] S. Laufer, H.-G. Striegel, K. Neher (Merckle GmbH),
DE 19842833 A1, 1998 (Chem. Abstr. 2000, 132, 210154).
[6] H. Lettau, Z. Chem. 1970, 10, 462.
IC50 Æ SEM [mm][a]
Inhibition [%] of
P450 isoforms[b]
Compound p38
TNF-a
IL-1b
2D6
3A4
[7] a) T. Ravindranathan, R. D. Wakharkar, A. B. Landge, Org. Prep.
Proced. Int. 1986, 18, 95 98; b) L. Testaferri, M. Tiecco, M. Tingoli, D.
Chianelli, M. Montanucci, Synthesis 1983, 751 755.
[8] a) G. Mloston, M. Celeda, G. K. S. Prakash, G. A. Olah, H. Heimgart-
ner, Helv. Chim. Acta 2000, 83, 728 738; b) I. J. Ferguson, K. Schofield,
J. Chem. Soc. Perkin Trans. 1 1975, 275 277; c) D. Wang, J. Haseltine,
J. Heterocycl. Chem. 1994, 31, 1637 1639.
SB 203580 0.29 Æ 0.03 (7) 0.59 Æ 0.09 (21) 0.037 Æ 0.006 (20) 73.1
76.6
87.1
28.3
16.5
28.8
ML 3163
4b
4.0 Æ 1.0
2.2 (1)
1.1 Æ 0.4 (4)
2.2 Æ 0.9
0.38 Æ 0.13 (4)
0.45 Æ 0.03
71.8
7.8
4c
4d
0.50 (1)
2.2 (1)
0.51 Æ 0.24 (4) 0.11 Æ 0.03 (4)
13.4
0.7
1.1 Æ 0.3
0.38 Æ 0.04
[a] Tests were carried out in duplicate, except where the number in brackets denotes
otherwise. SEM ˆ Standard error of measurement. [b] Results are from one
experiment each, carried out at a test-compound concentration of 10 mm in
phosphate buffer (pH 7.4) with DMSO (0.1%).
[9] M. Spatzenegger, W. Jaeger, Drug Metab. Rev. 1995, 27, 397 417.
favorable cell-penetration properties. In the whole-blood
assay, IC50 values (mm) for the most active derivatives 4b
(TNF-a: 5.6 Æ 0.95, IL-1b: 1.5 Æ 0.7), 4c (TNF-a: 0.51 Æ 0.24,
IL-1b: 0.11 Æ 0.03), and 4d (TNF-a: 5.1 Æ 0.4, IL-1b: 1.1 Æ 0.7)
were lower than those of lead compound ML 3163 (TNF-a:
20.3 Æ 4.8, IL-1b: 2.78 Æ 0.13), and close to the nanomolar
range. Finally, the most promising results came from the
toxicityscreen, in which 4b d (Table 1) onlymoderately
interacted with those P450 isoforms most important for drug
metabolism.[9] This profile gives 4b d a clear advantage over
both SB 203580 and ML 3163, and makes them strong
candidates for further development as anti-inflammatory
drugs.
Tetrafunctional Photoaffinity Labels Based on
Nakanishi×s m-Nitroalkoxy-Substituted
Phenyltrifluoromethyldiazirine**
Mohammed Daghish, Lothar Hennig,
Matthias Findeisen, Sabine Giesa, Frank Schumer,
Horst Hennig, Annette G. Beck-Sickinger, and
Peter Welzel*
Photoaffinitylabeling has been demonstrated to be a
remarkablyefficient method for studying the interactions of
biologicallysignificant compounds (ligands) with their target
macromolecules.[1] The method allows the identification of the
targets (for example, binding proteins) and, also the binding
domain within the target protein. An appropriate photo-
affinity-labeling compound should contain three structural
elements:
a) a ligand which directs the label to the binding site on the
protein,
b) a photolabile group for attaching to the protein,
c) an indicator that allows the identification of the labeled
peptides after enzymatic digestion of the labeled protein.
Received: November 6, 2001 [Z18173]
[1] J. C. Lee, A. M. Badger, D. E. Griswold, D. Dunnington, A. Truneh, B.
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Livi, J. R. White, J. L. Adams, P. R. Young, Nature 1994, 372, 739 746;
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Griswold, J. Pharmacol. Exp. Ther. 1996, 279, 1453 1461; d) J. L.
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Dancheck, A. J. Forsyth, D. S. Fletcher, B. Frantz, W. A. Hanlon, C. F.
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O×Keefe, J. Med. Chem. 1999, 42, 2180 2190.
[*] Prof. Dr. P. Welzel, M. Daghish, Dr. L. Hennig
Institut f¸r Organische Chemie
Universit‰t Leipzig
Johannisallee 29, 04103 Leipzig (Germany)
Fax : (‡49)341-9736-559
E-mail: welzel@organik.chemie.uni-leipzig.de
Dr. M. Findeisen, Dr. S. Giesa
Institut f¸r Analytische Chemie
Universit‰t Leipzig
Johannisallee 29, 04103 Leipzig (Germany)
F. Schumer, Prof. Dr. H. Hennig
Institut f¸r Anorganische Chemie
Universit‰t Leipzig
Johannisallee 29, 04103 Leipzig (Germany)
Prof. Dr. A. G. Beck-Sickinger
Institut f¸r Biochemie
Universit‰t Leipzig
Talstrasse 33, 04103 Leipzig (Germany)
[**] We are grateful to Mrs. R. Herold and Mrs. R. Oehme for technical
assistance. Financial support bythe Deutsche Forschungsgemein-
schaft, BC Biochemie GmbH, and the Fonds der Chemischen
Industrie is gratefullyacknowledged.
Supporting information for this article is available on the WWW under
http://www.angewandte.org or from the author.
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Such an indicator maybe a radioactive ligand or a
nonradioactive tag such as a biotin group. The latter has the
great advantage that it enables the labeling of the target
protein and labeled protein fragments to be demonstrated by
means of their interaction of biotin with avidin or streptavidin
and isolating them byaffinitychromatography. [2] Usually, the
probes have the general appearance of 1, that is, the photo-
labile group is attached to the ligand to which in turn the
biotin tag is connected in a linear arrangement. Thus, the
syntheses of these probes are linear and of limited efficiency.
viously[14]) to give 12 (Scheme 1B), which was fullycharac-
1
terized by H and 13C NMR spectroscopyas well as mass
[15]
spectrometry.
The photochemistryof the system was studied with model
compounds 4c and 7. Thus, experiments on an analytical scale
(0.76 mmolLÀ1 in methanol, mercuryhigh-pressure lamp,
monochromator, 350 nm) indicate (see the isosbestic points in
Figure 1) that 4c is cleanlyconverted into the methoxy
We have now developed the two photoaffinitylabeling
reagents 7 (Scheme 1A) and 18 (Scheme 3) which can
accommodate the photophore, here an aryl trifluoromethyl-
diazirine (a carbene precursor first introduced byBrun-
ner[3, 4]), the biotin tag, and the ligand attachment site
[5]
independently. Anydesired ligand can be coupled to the
free OH group of 7 and to the squaric ester function of 18 by
an appropriate method. In addition, these compounds have
two photolabile groupings, the diazirine moietywhich gen-
erates the carbene intermediate on irradiation at 350 nm[6]
and an alkyl m-nitrophenyl ether which undergoes a photo-
substitution reaction to yield the corresponding m-nitrophe-
nol on irradiation at the same wavelength in mildlybasic
Figure 1. UV/Vis spectroscopic control of the conversion of 4c into to 13.
[8]
solution.[7] As suggested byNakanishi and co-workers, the
derivative 13 (Scheme 2). On a preparative scale, irradiation
of 4c (0.7 mm in methanol, 108C, argon atmosphere, 350 nm,
Rayonet reactor) furnished the methoxy compound 13 in
70% yield (according to the 19F NMR spectrum of the
reaction mixture). After chromatographic purification, 13 was
fullycharacterized byNMR spectroscopyand mass spec-
trometry.
The aromatic photosubstitution bywhich the m-nitroalkoxy
derivatives are converted into the corresponding m-nitro-
phenols offers the verypromising opportunityto assist mass
spectrometric structural elucidation bysimplyexecuting the
photosubstitution in a mixture of H216O and H218O. We
performed the cleavage reaction of 13 in 0.01 molLÀ1 NaOH
in a 4:1 mixture of H216O and H218O. As Figure 2 shows the 4:1
ratio of H216O and H218O is nicelytranslated into a 4:1 ratio of
the intensities of the molecular ions of the substitution
products 14a and 14b. Thus, after a photoaffinitylabeling
experiment, all labeled peptides should be easilyrecognized
bydoublets with a mass difference of two in the mass spectra.
We also studied the photodegradation of photolabel 7 on an
analytical scale (1.34 mm in methanol) under the conditions
described above. The UV spectra are more complicated
latter reaction can be used to remove both the ligand and the
biotin tag from the peptide before mass spectrometric
sequencing to avoid complications in its mass spectrometric
analysis.
Amine 3 was prepared starting from dimethyl 5-hydroxy-
isophthalate (2) as described byNakanishi and co-workers
(Scheme 1A).[8] The oxidation of 3 with dimethyldioxirane
was alreadyknown to give the corresponding nitro compound
4a in a reported yield of 66%. We found the reaction to be
rather capricious. In our hands, the method of Krohn et al.[9]
(Zr(OtBu)4-mediated oxidation with tert-butyl hydroperox-
ide) gave far more reliable results. The yields of 4a were
uniformlyin the range of 80%. Oxidative degradation of 4a[10]
provided aldehyde 4b, and reduction of the latter with NaBH4
furnished primaryalcohol 4c in 84% yield. Isocyanate 6 was
[11]
obtained from (R)-malic acid (5) as described previously.
The coupling of 6 with primaryalcohol 4c gave urethane 9,
which reacted with the biotin derivative 8 (obtained from the
corresponding, commercial diamine and biotin activated with
N,N'-carbonyldiimidazole (CDI) in pyridine) to give 7
through amide formation and concomitant loss of the
1
protecting group. The H and 13C NMR spectra of 7 were
because of overlapping bands. The difference spectra (A(t)
À
fullyassigned byH,H COSY and 13C,1H COSY experiments.
The 19F NMR signal was found at d ˆ 12.65 ppm (CDCl3
solution, trifluoroacetic acid (TFA) d ˆ 0 ppm). The ESI mass
spectrum[12] showed the expected quasi molecular ion
peaks.[13] Compound 7 can be regarded as a broadlyapplicable
biotin-tagged photoaffinitylabel. We attached 7 to the
moenomycin derivative 11b (obtained from moenomycin A
bythe azo-coupling/Japp Klingemann route described pre-
A(tˆ0)) displayed in Figure 3 have been calculated to separate
the absorptions of the reaction site. Figure 3 again nicely
shows two isosbestic points indicative of a clean conversion of
7 into 15.
A second broadlyapplicable photoaffinityreagent based on
a m-nitroalkoxy-substituted phenyltrifluoromethyldiazirine
(Nakanishi diazirine) was obtained from aldehyde 4b and
amino compound 16 byreductive amination to give secondary
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Scheme 1. A) Synthesis of 7: a) 3, Zr(OtBu)4 (0.1 equiv), tert-butyl
hydroperoxide (3 equiv), molecular sieves (3 ä), CH2Cl2, 208C, 19 h,
flash chromatography (ethyl acetate/cyclohexane 2:8, Rf ˆ 0.7), 80%;
b) OsO4 (4% in H2O, 0.04 equiv), N-methylmorpholine-N-oxide
(1.2 equiv), acetone/water 8:1, 208C, 3 h, flash chromatography
(chloroform(methanol 30:1, Rf ˆ 0.2), NaIO4 on silica gel (obtained
from a 0.65 molLÀ1 aqueous solution, 14 equiv), RT, 24 h, flash
chromatography(chloroform/methanol 8:2, Rf ˆ 0.8), 74%; c) NaBH4
(1 equiv), THF/MeOH 4:1, 108C, 30 min, flash chromatography
(ethyl acetate/toluene/cyclohexane 3:4:3, Rf ˆ 0.4), 84%; d) 6
(1.5 equiv) in several portions, until TLC analysis showed complete
consumption of 4c, chloroform, RT, 5 d, flash chromatography(ethyl
acetate/cyclohexane 1:1, Rf ˆ 0.5), 88%; e) 8 and 9 (1.5 equiv), DME/
water 1:1, 08C, 2.5 h !RT, 12 h, flash chromatography(chloroform/
methanol 8:2, Rf ˆ 0.35), 65%. B) Synthesis of 12: 7 and 10 (1 equiv),
dimethylaminopyridine (0.9 equiv), pyridine, 08C, then water
(1 equiv), 11b (1.7 equiv) RT, 30 h, filtration through RP18 (acetoni-
trile/water 1:1, Rf ˆ 0.9), flash chromatography(methanol/chloroform
2:8, Rf ˆ 0.1, water/1-propanol 3:7, Rf ˆ 0.7, RP18 (acetonitrile/water
3:7), 10%.
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Figure 3. UV/VIS spectroscopic control of the conversion of 7 into
15, difference spectra A(t) À A(tˆ0)
.
compound 19, which was characterized byNMR spec-
[20]
troscopyand mass spectrometry.
Both 12 and 19 have been found to be antibiotically
active against a number of Staphylococcus aureus strains
(minimum inhibitoryconcentration (MIC): 4.8 Â 10À7
and 3.2 Â 10À7 molLÀ1, respectively)[21] although to a
lower degree than moenomycin A itself.[22]
In conclusion, we have developed two biotinylated
photoaffinitylabels based on Nakanishi×s diazirine that
can be attached to anysuitable ligand. The photo-
chemical removal of the ligand and biotin from labeled
peptides can be used to introduce a 16O/18O tag. This
should greatlyfacilitate identification of cross-linked
peptides bymass spectrometr.y Compounds 12 and 19 are
fullyequipped for photoaffinitystudies. In addition, theyhave
been found to be antibioticallyactive, and their use in the
photoaffinitylabeling of their target protein, the transglycos-
ylase domain of penicillin-binding protein 1b,[23] will be
reported in due course.
Scheme 2. Photochemical transformations.
Received: December 7, 2001 [Z18343]
[1] F. Kotzyba-Hilbert, I. Kapfer, M. Goeldner, Angew. Chem. 1995, 107,
1391 1408; Angew. Chem. Int. Ed. Engl. 1995, 34, 1296 1312; S. A.
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¬
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[3] J. Brunner, Annu. Rev. Biochem. 1993, 62, 483 514; T. Weber, J.
Brunner, J. Am. Chem. Soc. 1995, 117, 3084 3095.
Figure 2. Molecular ions of the photo-substitution product(s) of 13. Light
gray: reaction in H216O, black: reaction in 4:1 H216O/H218O.
[4] For a comparison of four photoactivatable probes, see P. J. A. Weber,
A. G. Beck-Sickinger, J. Pept. Res. 1997, 49, 375 383.
[5] See B. A. Gilbert, R. R. Rando, J. Am. Chem. Soc. 1995, 117, 8061
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[6] M. Nassal, Liebigs Ann. Chem. 1993, 1510 1523; Y. Ambroise F.
Pillon, C. Miokowski, A. Valleix, B. Rousseau, Eur. J. Org. Chem.
2001, 3961 3964, and references therein.
[7] E. Havinga, M. E. Kronenberg, Pure Appl.Chem. 1968, 16, 137 152.
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1194.
amine 17 in 60% yield (Scheme 3). Reaction of 17 with
diethyl squarate[16 18] provided squaric acid ester amide 18.[19]
As usual for squaric acid amides,[16] two 13C NMR signals
appeared for manycarbon atoms, thus indicating the presence
of two conformers. Compound 18 can be attached to any
suitable ligand with a primaryor secondaryamino group.
Thus, the reaction of 18 with the moenomycin derivative 11a
in a 1:1 mixture of water and methanol at pH 9 furnished
[9] K. Krohn, J. K¸pke, H. Rieger, J. Prakt. Chem. 1997, 339, 335 339.
2296
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COMMUNICATIONS
Scheme 3. Synthesis of 19: a) 4b and 16 (1.5 equiv), methanol, TFA (0.2 molLÀ1 in methanol, adjusting the pH to 7.0),
NaCNBH3 (1 equiv), RT, 32 h, flash chromatography(methanol/chloroform 1:9, Rf ˆ 0.1), 60%; b) 3,4-diethoxy-3-cyclo-
butene-1,2-dione (2.7 equiv), ethanol, RT, 19 h, flash chromatography(methanol/chloroform 2:10, Rf ˆ 0.5), 91%; c) 11a,
water/methanol 1:1, Et3N (adjusting the pH to 9), RT, 5 d, flash chromatography(1-propanol/water 7:2, Rf ˆ 0.5), 32%.
[10] M. Daumas, Y. Vo-Quang, L. Vo-Quang, F. Le Goffic, Synthesis 1989,
64 65.
[11] T. R¸hl, L. Hennig, Y. Hatanaka, K. Burger, P. Welzel, Tetrahedron
Lett. 2000, 4, 4555 4558; K. Burger, E. Windeisen, R. Pires, J. Org.
Chem. 1995, 60, 7641 7645; R. Pires, K. Burger, Synthesis 1996,
1277 1279, and references therein.
[18] A. Buchynskyy, U. Kempin, S. Vogel, L. Hennig, M. Findeisen, D.
M¸ller, S. Giesa, H. Knoll, P. Welzel, Eur. J. Org. Chem., in press.
‡
[19] C32H40F3N7O10S (771.77, 771.25), ESI MS: m/z ˆ 772.25759 [M‡H] ,
‡
calcd: 772.25822; 794.23993 [M‡Na] , calcd: 794.24017.
[20] C108H151F3N17O46PS (2543.50, 2541.94), ESI MS: m/z ˆ 1269.96424
([M À 2H]2À), calcd: 1269.96320; 864.30686 ([M À 3H]3À), calcd:
864.30637.
[12] FT-ICR-MS (Apex II, Bruker Daltonics, water/methanol).
‡
[13] C34H49F3N8O12S (850.86, 850.34), ESI MS: m/z ˆ 889.27644 ([M‡K] ),
[21] MIC values were determined bya serial twofold microdilution
‡
calcd: 889.27743; 873.30178 ([M‡Na] ), calcd: 873.30350.
method (Iso-Sensitest medium, Oxoid). A series of decreasing
[14] U. Kempin, L. Hennig, D. Knoll, P. Welzel, D. M¸ller, A. Markus, J.
van Heijenoort, Tetrahedron 1997, 53, 17669 17690.
concentrations of the compound under investigation was prepared
in the medium. For inoculations, 1 Â 105 cfumLÀ1 were used. The
MIC values were determined after 24 h at 378C (absence of visible
turbidity).
[15] C124H177F3N19O52PS2 (2917.95, 2916.09), ESI MS: m/z ˆ 971.02420
([M À 3H]3À), calcd: 971.02343.
[16] L. F. Tietze, M. Arlt, M. Beller, K.-H. Gl¸senkamp, E. J‰hde, M. F.
Rajewsky, Chem. Ber. 1991, 124, 1215 1221.
[22] S. Vogel, A. Buchynskyy, K. Stembera, K. Richter, L. Hennig, D.
M¸ller, P. Welzel, F. Maquin, C. Bonhomme, M. Lampilas, Bioorg.
Med. Chem. Lett. 2000, 10, 1963 1965, and references therein.
[23] See K. Stembera, A. Buchynskyy, S. Vogel, D. Knoll, A. A. Osman,
J. A. Ayala, P. Welzel, ChemBioChem, 2002, 3, 332 340.
[17] Review: A. Schmidt, Synthesis 1980, 961 994; L. F. Tietze, C.
Schrˆter, S. Gabius, U. Brinck, A. Goerlach-Graw, H.-J. Gabius,
Bioconjugate Chem. 1991, 2, 148 153; V. P. Kamath, P. Diedrich, O.
Hindsgaul, Glycoconjugate J. 1996, 13, 315 319; V. Pozsgay, E.
Dubois, L. J. Pannell, J. Org. Chem. 1997, 62, 2832 2864; J. Zhang, A.
¬
Yergey, J. Kowalak, P. Kovac, Carbohydr. Res. 1998, 313, 15 20.
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