L-phototryptophan

Article abstract of DOI:10.1002/ejoc.201201726

Phototryptophan is a new photoactivatable amino acid designed to be incorporated into tryptophan-containing peptides for photoaffinity labeling. The synthesis of phototryptophan starts from 6-bromoindole with the installation of an 1-azi-2,2,2-trifluoroethyl moiety, followed by microwave-assisted condensation with serine under mild conditions. Separation of the N-acetylated enantiomers was possible by employing Aspergillus aminoacylase. Differential scanning calorimetry showed that phototryptophan is thermally stable up to 100 °C. Boc-L-Phototryptophan was used to assemble a diazirine analog of the marine natural product hemiasterlin. L-Phototryptophan carries a photoactivatable diazirinyl substituent in the indole 6-position and could become interesting for photoaffinity labeling. The thermally stable compound survives conditions of peptide synthesis and was incorporated into a photoderivative of the marine natural product hemiasterlin. Copyright

Full text of DOI:10.1002/ejoc.201201726

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201201726
L
-Phototryptophan
Thomas Wartmann[a] and Thomas Lindel*[a]
Dedicated to the memory of Professor Richard Neidlein
Keywords: Chiral resolution / Amino acids / Photoaffinity labeling / Nitrogen heterocycles / Photochemistry
Phototryptophan is
a
new photoactivatable amino acid
Separation of the N-acetylated enantiomers was possible by
employing Aspergillus aminoacylase. Differential scanning
calorimetry showed that phototryptophan is thermally stable
up to 100 °C. Boc-L-Phototryptophan was used to assemble a
diazirine analog of the marine natural product hemiasterlin.
designed to be incorporated into tryptophan-containing
peptides for photoaffinity labeling. The synthesis of photo-
tryptophan starts from 6-bromoindole with the installation of
an 1-azi-2,2,2-trifluoroethyl moiety, followed by microwave-
assisted condensation with serine under mild conditions.
substituent, which could undergo 1,2-shift after the carbene
has been formed, which would result in an unreactive ole-
fin.
Introduction
Photoaffinity labeling (PAL) allows, in principle, the
identification of biological targets of natural products.[1,2]
A photoreactive moiety is installed in the candidate mole-
cule, and it becomes chemically reactive after irradiation.
Ideally, this should happen after the bioconjugate has
reached its final destination in the cell and should lead to
covalent binding with the target, followed by isolation and
analysis of the adduct. Benzophenone,[3] tetrafluorophen-
ylazide,[4] and aryl(trifluoromethyl)diazirine[5] moieties are
most frequently used in PAL experiments. Diazirines are
particularly suitable, because they are small and the re-
sulting carbenes are reactive.[6] However, the smaller the
molecule of interest is, the larger will be the relative struc-
tural change caused by appendage of those toolbox photo-
reactive units. The biological properties of the original mo-
lecule will thus be altered substantially.
l-Phototryptophan (3, Figure 1) with a diazirinyl sub-
stituent in the indole 6-position could become of use for
PAL in the case of tryptophan-containing natural products.
Candidate natural products include hemiasterlin (1) from
the marine sponges Hemiasterella minor[11] and Cymbastela
sp.[12] Compound 2 is a new photohemiasterlin derivative
that we chose as an example for the incorporation of 3.
In the absence of the 1-azi-2,2,2-trifluoroethyl moiety, the
compound has exhibited antimitotic activity (IC50 = 20 nm)
and cytotoxicity (IC50 = 40 nm), as shown by Andersen and
co-workers.[13]
In particular for natural product research, it would be
desirable to be able to choose from a larger variety of pho-
toreactive structures than that currently available, which
would mimic structures already present in the molecule
of question. However, not even for all proteinogenic amino
acids do photoreactive analogs exist. Moreover, with the
exception of photophenylalanine,[7] all other photoreactive
amino acids comprising l-photoleucine, rac-photoiso-
leucine/rac-photo-allo-isoleucine, l-photomethionine,[8] l-
photolysine,[9] and l-photoproline[10] share an α-hydrogen
[a] Institute of Organic Chemistry, TU Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany
Fax: +49-531-391-7744
E-mail: th.lindel@tu-braunschweig.de
Figure 1. l-Phototryptophan (3) as a building block for novel
photohemiasterlin 2, an analog of the cytotoxic marine natural
product hemiasterlin (1).
Homepage: www.oc.tu-bs.de/lindel
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201726.
Eur. J. Org. Chem. 2013, 1649–1652
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T. Wartmann, T. Lindel
SHORT COMMUNICATION
enhanced the electrophilicity of 13 by quaternization to tri-
methylalkylammonium iodide 14, followed by reaction with
15/NaH to the protected phototryptophan imine
(Scheme 1). A possible intermediate could be the methyl-
eneindolenine.[17] Hydrolysis of the imine provided rac-pho-
totryptophan ethyl ester (47% from 13), followed by sapon-
ification to rac-phototryptophan (rac-3).
Differing from their unsubstituted analogs, the diazir-
inyl-substituted indole derivatives are yellow and show
weak absorption maxima at 382–392 nm (ε = 2.6–2.8) in the
UV/Vis spectra (DCM/MeOH). The differential scanning
calorimetry (DSC) curves of rac-phototryptophan (rac-3)
and l-tryptophan show that rac-3 undergoes an exothermic
reaction between 100 and 150 °C, whereas tryptophan ex-
hibits an endothermic behavior at 280–300 °C (Figure 2).
Thus, phototryptophan is thermally stable under conditions
employed in chemical biology.
Results and Discussion
Deprotonation of 6-bromoindole[14] (4; KH, THF) fol-
lowed by Br–Li exchange (tBuLi, CAUTION!)[15] and
quenching with the trifluoroacetic acid (TFA)-derived
Weinreb amide afforded 6-trifluoroacetylindole (5, 56%;
Scheme 1). However, it proved to be advantageous to pro-
ceed via the N-silylated indole, which was synthesized from
4 without workup; this species was then trifluoroacetylated
to 6 by treatment with CF3CO2Et (80%). Reaction of 6
with NH2OH·HCl in pyridine/EtOH, followed by tosyl-
ation provided tosyloxime 8, which was converted directly
into diaziridine 10 by reaction with liquid ammonia
(–70 °C) to avoid loss of material by chromatography. Start-
ing from unprotected indole 5, the analogous sequence af-
forded N-free diaziridinylindole 9, albeit in lower yield. Oxi-
dation to stable diazirines 11 and 12 was possible with
pyridinium chlorochromate (PCC) and I2/NEt3, respec-
tively. Desilylation of 12 to 11 went smoothly, and even af-
ter several months of storage at –18 °C, no decomposition
of 11 could be observed.
Figure 2. Analysis of l-tryptophan and rac-phototryptophan (rac-
3) by DSC.
Upon irradiation of a solution of N-silylated photoindole
12 in MeOH at λmax = 350 nm (Rayonet), no reaction took
place. However, at λmax = 300 nm diazirine 12 was con-
verted into methyl ether 16 (66% isolated yield after 5 h,
Scheme 2). Interestingly, the use of a DCM/MeOH (11:1)
solvent mixture allowed the photolabeling reaction to be
carried out even at λmax = 365 nm (UV-A lamp, Rayonet).
N-Acetyl-rac-phototryptophan (rac-17, 10 mm, synthesis
Scheme 1. Synthesis of rac-phototryptophan (rac-3) via photo-
gramine (13).
Aminomethylation of 11 provided photogramine 13. Un-
der Somei–Kametani conditions[16] in the presence of
P(nBu)3 at 80 °C, compound 13 decomposed upon reaction
Scheme 2. Photoreaction of diazirinylindoles 12 and rac-17 with
with glycine-derived benzophenonimine 15. Therefore, we MeOH to afford methyl ethers 16 and 18, respectively.
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l-Phototryptophan
Scheme 3) afforded methanol adduct 18 (60%, 1 h). A pos- (Scheme 4). The coupling conditions proved to be superior
sible reason is the stabilization of the singlet carbene by to the use of bromotripyrrolidinophosphonium hexafluoro-
DCM, which can lead to an increase in the life time by a phosphate (PyBrop), which was originally used by An-
factor of more than 20.[18]
dersen and co-workers for the coupling of Boc-protected
tryptophan and 23.[13] Saponification and removal of the
Boc group provided photohemasterlin 2 (35%).[20]
Scheme 4. Application of l-(–)-phototryptophan [(S)-3] in the syn-
thesis of photohemiasterlin (2) (HOBt: N-hydroxybenzotriazole;
EDC: N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydro-
chloride; NEM: N-ethylmorpholine).
Conclusions
In summary, we have developed an efficient synthesis of
optically pure l-(–)-phototryptophan [(S)-3] and its Boc-
protected version 22.[21,22] d-(–)-N-Acetylphototryptophan
is also accessible in high optical purity, and it should be
able to undergo conversion into d-(+)-phototryptophan. A
DSC experiment revealed that phototryptophan is stable
below 100 °C. N-Boc-l-(–)-Phototryptophan (22) can be
used under standard conditions of peptide synthesis, as
shown by the synthesis of photohemiasterlin (2). Irradiation
experiments showed that phototryptophan is indeed photo-
reactive, and this leads to the formation of ether with meth-
anol at wavelengths up to 365 nm. Aspergillus aminoacylase
tolerates the presence of the 1-azi-2,2,2-trifluoroethyl moi-
ety at the indole 6-position. The enzyme itself could now be
among the first candidates for PAL with phototryptophan
derivatives. Furthermore, it may also become of interest to
incorporate 3 onto the protein side.
Scheme 3. Enantioselective synthesis of l-phototryptophan [(S)-3]
and d-N-acetylphototryptophan [(R)-17] by enzymatic hydrolysis.
Enantiomeric excess was determined by HPLC analysis of methyl
esters (R)-21 and (S)-21.
For the synthesis of enantiomerically pure l-phototryp-
tophan [(S)-3] and N-acetyl-d-phototryptophan [(R)-17] we
envisaged enzymatic deacetylation of rac-N-acetylpho-
totryptophan (rac-17) and, therefore, had to avoid the
benzophenone intermediate. However, condensation of di-
aziridinylindole 11 with serine/Ac2O-HOAc (via the az-
lactone) at 70 °C led to decomposition. Interestingly, the
reaction was successful under carefully chosen, mild micro-
wave conditions; the temperature was kept at about 45 °C
(IR thermometer) by employing pulses of weak power over
a time period of 2 h. rac-N-Acetylphototryptophan (rac-17)
was isolated in 50% yield (Scheme 3).
Enzymatic resolution of rac-17 was possible by treatment
with Aspergillus aminoacylase (specific activity 31 units/mg)
in the presence of catalytic amounts of CoCl2·6H2O (pH 7).
After reverse-phase chromatography, l-(–)-phototrypto-
phan [(S)-3, 24%; [α]2D3 = –24.5, c = 0.44, MeOH] and d-
(–)-N-acetylphototryptophan [(R)-17, 31%; [α]2D3 = –22.8, c
= 0.51, MeOH] were obtained. Analysis of the enantiomeric
purity was performed on the level of methyl esters (S)-21
(98%ee; [α]2D3 = +43.6, c = 0.55, CHCl3) and (R)-21
(93%ee; [α]2D3 = –40.7, c = 0.55, CHCl3), which were ob-
tained by reaction with TMSCHN2 followed by HPLC on
a Chiralcel OJ column.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental procedures; 1H NMR and 13C NMR spectra of
compounds 5–24; DSC measurements of rac-3, 11, 13, and rac-17;
HPLC analysis of (R)-21 and (S)-21.
Acknowledgments
Financial support of this research by the Niedersächsische Techni-
sche Hochschule (NTH, bottom-up project) is gratefully acknowl-
edged. We also thank Merck KGaA (Darmstadt, Germany) for the
generous gift of chromatography materials. BASF Group (Lud-
wigshafen, Germany) and Honeywell Specialty Chemicals Seelze
GmbH (Seelze, Germany) are thanked for the donation of solvents.
For the synthesis of photohemiasterlin 2, l-phototrypto-
phan [(S)-3] was Boc-protected to 22 and coupled with
peptide 23[13] (EDC/HOBt)[19] to afford Boc ester 24
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Published Online: February 1, 2013
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