Water compatible photoarylation of amino acids and peptides

Article abstract of DOI:10.1002/chem.201402959

A novel photoarylation of amino acids and peptides is described, which tolerates the presence of water. Irradiation of Boc-protected amino acids in the presence of N-protected 2-azidobenzimidazoles leads to selective arylation of carboxy termini or side chains. The new reaction also works for peptides. Irradiation of the nonapeptide H-SPSYVYHQF-OH also resulted in selective arylation of the tyrosine side chains, as indicated by ESI-MS/MS fragmentation. Chemo- and regioselectivity could add the title reaction to the repertoire of photoaffinity labeling methods.

Full text of DOI:10.1002/chem.201402959

DOI: 10.1002/chem.201402959
Communication
&
Bioorthogonal Chemistry
Water Compatible Photoarylation of Amino Acids and Peptides
Alex Sudakow, Uli Papke, and Thomas Lindel*[a]
reactions would occur with the reactive nitrene intermediate
formed on irradiation.
Abstract: A novel photoarylation of amino acids and pep-
tides is described, which tolerates the presence of water.
Irradiation of Boc-protected amino acids in the presence
of N-protected 2-azidobenzimidazoles leads to selective
arylation of carboxy termini or side chains. The new reac-
tion also works for peptides. Irradiation of the nonapep-
tide H-SPSYVYHQF-OH also resulted in selective arylation
of the tyrosine side chains, as indicated by ESI-MS/MS
fragmentation. Chemo- and regioselectivity could add the
title reaction to the repertoire of photoaffinity labeling
methods.
Scheme 1. Questions regarding a possible photoarylation of peptides with
2-azidobenzimidazoles.
Chemoselective functionalization of peptides and proteins is of
key importance for biotechnology and chemical biology.[1]
Most methods focus on reacting cysteine[2] and lysine[3] side
chains. It has also become possible to functionalize tyrosine[4]
and tryptophan[5] via transition-metal catalysis. Photoreactions
of peptides are particularly desirable, because light is bioor-
thogonal. Here, addition of thiyl radicals to alkenes has allowed
photochemical immobilization of proteins.[6] Photoreactions
have also been applied to genetically engineered proteins con-
taining non-proteinogenic amino acids.[7] Benzophenones, phe-
nylazides, and diazirines[8] react with limited chemoselectivity.[9]
It can be stated that there is still room for improvement, ideal-
ly involving new chemistry.
In this communication, it is demonstrated for the first time
that carboxy groups of amino acids and small peptides can be
photoarylated in the presence of water.
At the beginning it was to be investigated how low we
could go with the concentration of the valuable acid reaction
partner, which had been a solvent component in the case of
HOAc. Thus, we analyzed the reaction of Boc-Asp-OBn (1,
Scheme 2), which was irradiated in the presence of azide 2.[11]
Recently, we reported that irradiation of N-(p-fluorobenzyl)-
2-azidobenzimidazole in AcOH/CH2Cl2 afforded the 2-amino-6-
acetoxybenzimidazole in high yield and perfect regioselectivi-
ty.[10] Probably, the initially formed nitrene intermediate
(Scheme 1) undergoes ring opening to a non-symmetrical di-
azaxylylene which is protonated at the more basic imine nitro-
gen, followed by nucleophilic attack of acetate at the b-posi-
tion of the iminium ion, ring closure to the 2-iminoimidazole,
and rearomatization via proton shift. The transfer of that reac-
tion to the case of peptides with their various functional
groups would be very interesting. The presence of water had
to be tolerated, which was unclear, and the stoichiometry of
the photoarylation had to be investigated (Scheme 1). It was
also unclear which yields would be achievable and which side
Scheme 2. Photoarylation of Boc-Asp-OBn and yields depending on the
equivalents of carboxylic acid.
In the presence of more than 5 equiv of 1, photoarylation
yields around 80% were achieved, referred to azide 2. Use of
3 equiv of 1 gave 70%, while 1 equiv still afforded 33% of aryl-
ester 3. The amino acid core structure remained intact and the
Boc group in place. We decided to employ 4 equiv of carboxyl-
ic acid in the following experiments.
[a] A. Sudakow, Dr. U. Papke, Prof. Dr. T. Lindel
Institute of Organic Chemistry, TU Braunschweig
Hagenring 30, 38106 Braunschweig (Germany)
Fax: (+49)531-391-7744
We were pleased to find that the Boc-protected amino acids
alanine, valine, leucine, phenylalanine, and glycine with non-
functionalized side chains (4 equiv) reacted smoothly at the
E-mail: th.lindel@tu-bs.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402959.
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Communication
carboxy group in 54–79% isolated yield (based on the azide
component) after removal of excess amino acid (aq. NaHCO3)
and normal phase chromatography (Scheme 3). Boc-Ser-OH
was arylated at the carboxy terminus in the good yield of
72%. The aliphatic hydroxy group was tolerated. The reaction
with Boc-Tyr-OH at longer wavelength (370 nm) gave the ester
in lower 33% yield, accompanied by formation of 1-(p-fluoro-
benzyl)-2-aminobenzimidazole (29%).[10]
Scheme 4. Products obtained on irradiation Boc-Lys-OH in the presence of
N-methylated azidobenzimidazole 13. R=p-fluorobenzyl.
6-Chlorination also occurred on reaction of 2 with hydrochloric
acid in methanol.[10]
Irradiation of doubly Boc-protected cystine (4 equiv,
Scheme 5) and quenching with pyrrolidine afforded pyrrolidine
carboxamide 17 as major product (27%). In addition, bis
adduct 18 (22%) was isolated, presumably formed after com-
peting arylation of the disulfide sulfur by intermediate 19, fol-
lowed by nucleophilic attack of pyrrolidine at the sulfonium
salt. However, we were not able to detect a sulfur containing
benzimidazole-derived reaction product. 6-Hydroxybenzimida-
zole 11, formed by aminolysis of the intermediate aryl ester,
was isolated in 83% yield.
Scheme 3. Photoreaction of azidobenzimidazole 2 with eight Boc-protected
proteinogenic amino acids. Serine derivative 9 was obtained in CH2Cl2/
tBuOH (1:24). For the synthesis of tyrosine derivative 10, it was irradiated at
lmax =370 nm in CH2Cl2/tBuOH (1:9).
We could prove sulfur arylation for the case of Boc-Met-OH,
where irradiation afforded four major products (Scheme 5). Be-
sides the expected Boc-carboxamide 20 (17%) and 11 (14%)
we isolated Boc-homoserine carboxamide 21 (28%) and 2-
amino-6-(methylthio)benzimidazole 22 (20%). Thus, the thio-
ether moiety is a competitive nucleophile undergoing a reac-
tion similar to cyanogen bromide cleavage.
Chromatography of more polar photoarylation products re-
quired the use of protic mobile phases, which led to substan-
tial loss by hydrolysis or methanolysis of the aryl ester with for-
mation of 2-amino-6-hydroxybenzimidazole (11). We found
that addition of pyrrolidine (8–12 equiv based on azide 2) to
the reaction mixture immediately after irradiation afforded
stable carboxamides via acyl transfer after 24–72 h. In the case
of Boc-His-OH, quenching with pyrrolidine led to carboxamide
12 (30%, Scheme 3). The imidazole side chain did not react.
Importantly, we realized that photoarylation with 2 is water
tolerant.
In the case of Boc-Trp-OH we were not able to isolate de-
fined products. However, tryptophan-containing peptides and
proteins have been photolabeled after irradiation with UV
light,[13] meaning that tryptophan could still be tolerated under
photoarylation conditions when being part of a longer pep-
tide.
Dipeptide Boc-Ile-Thr-OH (23) and tripeptide Boc-Asn-Ile-Thr-
OH (24)[14] served as first examples containing peptide bonds
(Scheme 6). We were pleased to observe that photoarylation
yields of 66% (25) and 75% (26) were obtained, respectively,
with the threonine and asparagine side chains and the peptide
bonds remaining intact. This not only proves that peptide
bonds are inert to our reaction conditions, but also indicated
that peptides may react in a cleaner manner than the Boc-pro-
tected amino acids themselves.
Reaction of Boc-Lys-OH with N-benzylated azide 2 was un-
successful, even in the presence of phosphoric acid. We had
observed earlier that the presence of phosphoric acid leads to
incorporation also of neutral nucleophiles such as MeOH.[10]
However, the N-methyl analogue 13[12] (Scheme 4) reacted in
the presence of phosphoric acid affording 2-amino-1-methyl-
benzimidazole (14, 18%), e-lactam 15 (20%, by intramolecular
aminolysis), and adduct 16 (52%). With Boc-Arg-OH hydrochlo-
ride chlorination of the benzimidazole 6-position occurred
(56%) instead of arylester formation, probably because the
guanidinium side chain and the carboxy terminus remained
protonated, making chloride the best nucleophile in solution.
In the case of longer peptides, relative rates of reaction of
different functional groups will still depend on the precise
structure. This is illustrated in the following example. The
murine tumor-associated antigen AH1 (27, H-SPSYVYHQF-OH,
Scheme 7)[15] is a nonapeptide which contains two phenol moi-
eties (Tyr4, Tyr6), two aliphatic hydroxy groups (Ser1, Ser3) and
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Scheme 6. Photoarylation of small peptides (lmax =300 nm, Rayonet, 15 mm,
2 h). a) Boc-Ile-Thr-OH (4 equiv), tBuOH/CH2Cl2 (5:1); b) Boc-Asn-Ile-Thr-OH
(4 equiv), tBuOH/CH2Cl2/H2O (10:4:1).
Scheme 7. Correlation of observed mass peaks in the fragment spectrum of
monolabeled nonapeptide AH1 (27)-derived peptides with expected b and y
fragment series (ESI+, HCD, Orbitrap). Fragments in italics carry the 2-ami-
nobenzimidazole unit. For details, see the Supporting Information.
alone it cannot be predicted at present, whether a phenolic
OH or a carboxy group will react faster in a given amino acid
sequence. However, all other functional groups present in 27
remained inert, which is important.
Scheme 5. Photoarylation of Boc-Met-OH and Boc2-cystine via two compet-
ing pathways (a,b), followed by quenching with pyrrolidine (R=p-fluoroben-
zyl; lmax =300 nm, 15 mm, 2 h, RT, CH2Cl2/tBuOH (2:1) in the case of Boc2-cys-
tine, CH2Cl2 in the case of Boc-methionine).
In summary, we have demonstrated for the first time that it
is possible to chemoselectively photoarylate native peptides.
The title reaction may become a new selective tool adding to
the repertoire of peptide functionalization. Its photochemical
character and tolerance to water makes the photoarylation
bioorthogonal.
one histidine side chain, in addition to the free amino and car-
boxy termini. Photoarylation of the double TFA salt of 27 with
azide 13 (3 equiv, 1.02 mm, lmax = 370 nm, 6 h) under argon in
H2O/tBuOH/CH2Cl2 (12:12:1) was successful. ESI-MS analysis of
the reaction mixture led to the identification of aminobenzimi-
dazole 14, adduct 16, unreacted peptide 27, and mono and bi-
sarylated peptides. We were able to assign 25 of the observed
mass peaks to fragments following known fragmentation path-
ways for protonated peptides (Scheme 7).[16]
Acknowledgements
Financial support of this research by the Deutsche Forschungs-
˘
gemeinschaft is gratefully acknowledged. Dr. Rodica S¸oldanes-
cu is thanked for the preparation of tripeptide 24. Dr. Werner
Tegge (HZI Braunschweig) is thanked for a sample of nonapep-
tide 27.
Surprisingly, the carboxy terminus had not been arylated. In-
stead, the fragmentation patterns indicate that, interestingly,
photoarylation of 27 had occurred at both tyrosine side chains.
Fragments indicating functionalization at other positions were
not observed. A control irradiation experiment with N-acetyl-
tyrosinamide (Ac-Tyr-NH2) in the presence of photolabel 13
showed that the phenol moiety alone is not photoarylated.
Probably, the precise structure of nonapeptide 27 is responsi-
ble for the altered reactivity. We conclude that from a sequence
Keywords: arylation · bioorthogonal reactions · nitrenes ·
peptides · photochemistry
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Published online on July 14, 2014
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