A trifunctional reagent for photoaffinity labeling

Article abstract of DOI:10.1016/S0040-4039(00)00699-7

A photolabel, a biotin tag, and a moenomycin ligand have been attached orthogonally to the three functional groups of isoserine to provide compound 13 that is to be used in affinity labeling of penicillin binding protein 1b. The urethane group in 13 can b

Full text of DOI:10.1016/S0040-4039(00)00699-7

Tetrahedron Letters 41 (2000) 4555±4558
A trifunctional reagent for photoanity labelingy
Thomas Ruhl,a Lothar Hennig,a Yasumaru Hatanaka,b Klaus Burgera
and Peter Welzela,*
aUniversitat Leipzig, Institut fur Organische Chemie, Johannisallee 29, D-04103 Leipzig, Germany
È
È
bResearch Institute for Wakan-yaku, Toyama Medical and Pharmaceutical University Sugitani 2630,
Toyama 930-01, Japan
Received 6 April 2000; accepted 27 April 2000
Abstract
A photolabel, a biotin tag, and a moenomycin ligand have been attached orthogonally to the three
functional groups of isoserine to provide compound 13 that is to be used in anity labeling of penicillin
binding protein 1b. The urethane group in 13 can be cleaved with n-butylamine in methanol or water.
# 2000 Elsevier Science Ltd. All rights reserved.
Photoanity labeling can provide important information about binding sites of enzyme±substrate
or enzyme±inhibitor complexes.1 After photolabeling the enzyme is usually digested enzymatically
and the labeled fragments are isolated and submitted to structural analysis. Biotin-labeling is
a powerful method for identifying labeled protein fragments and isolating them by anity
chromatography based on the strong interaction of biotin with either avidin or strepavidin.2 Thus
a carbene-generating photoprobe3 of type 1 has been prepared and used for labeling the acceptor
binding site of a galactosyltransferase (Scheme 1).4 Recently, Nakanishi and co-workers discussed
the problem of elucidating the structures of the labeled peptides by tandem MS without separation
of the mixture and they proposed that it would be desirable to remove both the ligand and the
biotin tag from the peptide before MS sequencing in order to avoid complications in MS analysis.
Based on these considerations they designed a bifunctional photoanity probe of type 2. These
compounds have two photolabile groupings: (i) the diazirine moiety which generates a carbene
intermediate on irradiation at 350 nm and (ii) the alkyl m-nitrophenyl ether which on irradiation at
365 nm undergoes a photosubstitution reaction in mildly basic solution to yield the corresponding
m-nitrophenol. The latter reaction can be used to remove the ligand-biotin part before MS
sequencing.5 We were interested in a compound of type 3 for anity labeling of the transglycosylase
module of penicillin-binding protein 1b. This enzyme catalyzes one of the last steps of the biosynthesis
of bacterial cell wall peptidoglycan.6
* Corresponding author. E-mail: welzel@organik.chemie.uni-leipzig.de
y
Dedicated with best wishes to Professor Horst Kessler on the occasion of his 60th birthday.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00699-7

4556
Scheme 1.
Scheme 2.

4557
For our purposes 2 seemed to have a number of disadvantages. The synthesis of the aryl tri-
¯uoromethyl diazirine moiety takes more than 15 steps. Furthermore, it seemed almost impossible
to couple the biotin tag to the moenomycin ligand without losing the binding capacity to the
enzyme.6 We devised, therefore, the new photolabeling system 3 based on a trifunctional isoserine
sca€old which can accommodate the aryl tri¯uoromethyl diazirine, the biotin tag, and the ligand
independently at its three di€erent functional groups (see 3). The cleavage site is the urethane
grouping that can be cleaved with butylamine in methanol or water (vide infra).
Based on these considerations we decided to prepare compound 10, the synthesis of which
makes use of the protection/activation strategy for a-amino and a-hydroxy acids that was developed
by Burger and co-workers.7
Scheme 3.

4558
Thus, treatment of malic acid with hexa¯uoroacetone gave 5, the azide of which was submitted
to a Curtius rearrangement to provide 6 as described previously7 (Scheme 2). Reaction of 6 with
Hatanaka's anity label 78 in CHCl3 solution and subsequent puri®cation by FC (petroleum
ether:ethyl acetate, 4:1) furnished urethane 8 in 71% yield (based on 7). Reaction of 8 with the
commercial biotin derivative 9 (for R see formula 10, 1 equiv.) in 1:4 dimethoxyethane:water (15 h
at 20ꢀC) occurred as expected, i.e. by amide formation and concomitant loss of the protecting
1
group to provide 10 in 50% yield after lyophilization and FC (CH2Cl2:methanol, 4:1). The H
and 13C NMR spectra of 10 have fully been assigned using H,H COSY, 13C,1H HMBC and
13C,1H HMQC. The 13C chemical shifts (CDCl3) around the CF3 groups are as follows:
ꢀ=122.57 (1JC,F=275 Hz, CF3), 29.66 (2JC,F=40.5 Hz, C-CF3).
Compound 10 is a general reagent to which any ligand can be attached in a suitable way via the
free OH group. We chose the bifunctional linker 4-isothiocyanato benzoyl chloride (11) to couple
the moenomycin-derived amine 126 (the rest of the moenomycin structure is apparent from formula
13) to 10 (Scheme 3). Thus, a mixture of 10, 11, and Steglich's base in dry pyridine was stirred at
^15ꢀC for 72 h. To the reaction mixture a solution of 12 (0.8 equiv.) in dry DMF was added and
the mixture was stirred at 20ꢀC for 55 h. Then water was added and solvents were removed by
lyophilization. Subsequent FC (1-propanol:water 4:1) provided 13 in 41% yield [19F NMR:
ꢀ=12.84 (external CF3COOH), 31P NMR: ꢀ=^2.19, external H3PO4)].
Treatment of 7 and 13 with butylamine in methanol (re¯ux) gave identical products as indicated
by TLC (petroleum ether:ethyl acetate, 2:1), and the same observation was made when the reaction
was performed in water (70ꢀC). These results indicate, that indeed the urethane grouping can be
cleaved under these conditions to release both the ligand and the biotin moiety. Work is in progress
now to make use of compound 13 in the sense that was discussed in the introductory section.
Acknowledgements
We wish to thank A. Buchynskyy for a sample of 12 and K. Richter for skilled assistance.
Financial support by the Deutsche Forschungsgemeinschaft (Innovationskolleg `Chemisches Signal
und biologische Antwort') and the Fonds der Chemischen Industrie is gratefully acknowledged.
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