Traceless Staudinger acetylation of azides in aqueous buffers

Article abstract of DOI:10.1016/j.bmc.2013.02.045

In this paper, we demonstrate the applicability of water-soluble p-dimethylaminoethyl substituted phosphinomethanethiol in acetyl transfer reactions by the traceless Staudinger ligation with unprotected *-azido lysine containing peptides in aqueous buffer systems. Additionally, we present an improved synthesis pathway for the water-soluble phosphinothiol linkers requiring less steps in a comparable overall yield in comparison to previously published protocols.

Full text of DOI:10.1016/j.bmc.2013.02.045

Bioorganic & Medicinal Chemistry 21 (2013) 3465–3472
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Traceless Staudinger acetylation of azides in aqueous buffers
c,d,
Sylwia Sowa a, , Michaela Mühlberg b,c, , K. Michal Pietrusiewicz a, , Christian P. R. Hackenberger
⇑
⇑
a Maria Curie-Sklodowska University, Department of Organic Chemistry, ul. Gliniana 33, 20-614 Lublin, Poland
b Freie Universität Berlin, Institut für Chemie und Biochemie, Takustr. 3, 14195 Berlin, Germany
c Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Roessle Str. 10, 13125 Berlin, Germany
d Humboldt Universität zu Berlin, Institut für Organische und Bioorganische Chemie, Institut für Chemie, Brook-Taylor-Str. 2, 12489 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 7 March 2013
In this paper, we demonstrate the applicability of water-soluble p-dimethylaminoethyl substituted phos-
phinomethanethiol in acetyl transfer reactions by the traceless Staudinger ligation with unprotected
e
-azido lysine containing peptides in aqueous buffer systems. Additionally, we present an improved
Keywords:
Traceless Staudinger ligation
Acetylation
Chemoselective
Protein modification
Water-soluble
Azides
synthesis pathway for the water-soluble phosphinothiol linkers requiring less steps in a comparable
overall yield in comparison to previously published protocols.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
In order to access proteins that are posttranslationally modified
at selected lysine residues, several strategies have been developed
The human genome has about 20,000 genes to code all different
proteins that control our daily life on a cellular level.1 Since this
number is not sufficient to undertake all the different functions
in the body, nature employs, in addition to RNA splicing events,
several enzymatic posttranslational modifications, such as acetyla-
tion, prenylation, glycosylation, methylation and phosphorylation,
which add additional levels of complexity to the protein level.2
These apparently small molecular changes can have a big impact
on the protein’s specific function by influencing its overall struc-
ture and can lead to activation or deactivation of enzymes or other
cellular processes.2 Recently, many proteins have been discovered
in the cytoplasm of eukaryotes and prokaryotes that carry different
lysine side chain modulations, such as acetylation or methylation,
but their specific role is only partially understood.3 For example,
histone proteins—found in the nuclei as part of the chromatin—ap-
ply these posttranslational modifications on lysines to alter the
chromatin’s nucleosomal structure. These changes were shown to
be critical in epigenetic processes such as gene repression or
activation controlling the transcription of DNA and thereby the
evolution of life.3
by chemical biologists over the last years. These approaches in-
clude the use of Native Chemical Ligation (NCL) for the introduc-
tion of lysine derivatives in the N-terminal histone tail,4
enzymatic lysine acetylation5 and amber suppression that enables
selective incorporation of acetyl lysine by employing an
aminoacyl-tRNA synthetase and tRNACUA pair created by direct
evolution.6 Another common approach for the access of site-
specifically modified biopolymers is the introduction of non-native
functional groups, so-called bioorthogonal reporters, into biopoly-
mers that can chemically react in a selective fashion without
protection of distal naturally occurring functional groups.7 Recent
studies often involve the application of an azide group as to date,
several well explored methods for its incorporation into a biomol-
ecule exist.8 The ready access to proteins carrying unnatural func-
tional groups is complimented by the engineering of several
synthetic transformations during the last years that use proteins
as substrates for mild and chemoselective organic reactions.7 In
addition to using these reactions for the attachment of functional
modules, such as purification tags or fluorescent labels, several
studies were devoted to mimic enzymatic protein modifications
by chemical transformations.9 For purpose of installing acetylated
lysine side chains in proteins, a thiol-ene based labeling strategy
that enables selective conversion of cysteines to acetyl lysine
derivatives on proteins, was published recently,10 which is only
limited by the presence of additional cysteines in the protein.
⇑
Corresponding authors. Fax: +49 (0)30 947931.
E-mail addresses: kazimierz.pietrusiewicz@poczta.umcs.lublin.pl (K.M. Pietru-
siewicz), hackenbe@fmp-berlin.de (C.P.R. Hackenberger).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.045

3466
S. Sowa et al. / Bioorg. Med. Chem. 21 (2013) 3465–3472
O
O
O
R'
R'
N
R
S
PAr2
+
N3 R'
R
N
H
R
S
PAr2
2
1
aza-ylide 3
4
R'=Gly-OH
Gly-NH2
R = Ac
Gly-Ac
Ala-Ac
Gly-NHBn
N
N
n
N
OH
P
SH
P
SH
SH
P
P
n = 1,2
n
N
N
N
5
6
7
8
Scheme 1. Traceless Staudinger ligation with water-soluble phosphines and different phosphine linker systems 5–8.
In this paper, we aim to develop an alternative for the purpose
of installing acetylated lysine side chains in unprotected peptides
by a chemical modification reaction. We chose the traceless Stau-
2. Results and discussion
2.1. Phosphine synthesis
dinger ligation for the conversion of
e-azido-lysine residues
(Scheme 1).11 The traceless Staudinger ligation—first developed
independently by Raines12 and Bertozzi13—is based on the Stau-
dinger reaction,14 in which an azide 1 reacts with a phosphine 2
via a four-membered cyclic transition state to an aza-ylide 3. In-
stead of a P@N bond hydrolysis in the Staudinger reduction to yield
an amine and a phosphine oxide, a (thio-)ester was introduced as
an electrophilic trap to enable an intramolecular shift and amide
bond formation to 4 under the release of phosphine oxide.15
Among the few designed phosphine linkers, (diphenylphosphino-
)methanethiol (5) has shown great potential and practical applica-
bility towards small molecules,16 but its main shortcoming was its
weak solubility in aqueous media required for peptides and pro-
teins. The successive efforts of Raines and his group have resulted
in a first family of water-soluble phosphines and opened up new
possibilities for ligations in aqueous systems.17 It was shown that
compounds 6 and 7 bearing dimethylamino-substituents give bet-
ter conversions than negatively charged derivatives, such as car-
boxylic acids. In addition, studies on coulombic effects depending
on the position of the dimethylamino-groups on the two aryl sub-
stituents showed a positive impact of meta-positioned amino
groups in 7.17 Subsequently, Marx has presented water-soluble
phosphine 8 possessing a phenol core instead of a thiomethylene
moiety.18 Notably, none of the water-soluble variants has to the
best of our knowledge been subjected to traceless Staudinger liga-
tions on more complex unprotected peptides or proteins.19
Our synthesis started with a n-butyllithium mediated metal–
halogen exchange on commercially available 1,4-dibromobenzene
9 and substitution of the formed lithium reagent with ethylene
epoxide (Scheme 2). Distillation yielded 85% of pure 4-bromophe-
nylethyl alcohol 10. The alcohol was converted into mesylate 11 in
96% yield after crystallization and then transformed into amine 12.
By the addition of another portion of dimethylamine (2 equiv) and
prolonged reaction times, the conversion of the reaction could be
increased from 67% to 97%. The resulting 4-(2-dimethylamino-
ethyl)bromobenzene 12 was converted into the Grignard reagent
and reacted with diethyl phosphite. Phosphine oxide 13 could be
isolated in 67% yield. Subsequently 13 was reduced by reaction
with an excess of diisobutylaluminum hydride (DIBAL-H). The free
phosphine and the amino groups were directly complexed with
BH3 to give the secondary phosphine borane 14 in 46% yield. This
one-pot reduction protocol proved to be the most demanding syn-
thetic transformation in this route due to the sensitivity of 14 dur-
ing isolation. The last step was performed by alkylation of 14 with
acetylthiomethyl bromide. The newly developed one-step proce-
dure yielded 60% of the desired product 15, which corresponds
to a similar overall yield of 15%, which was reported by Raines
et al. in 2007.17a,20 In comparison to the previously published syn-
thesis, we reduced the number of reaction steps from seven to five
and could increase the yield from 14 to 15 from 46% to 60%.17a
In this paper, we first present a modified synthetic route to
2.2. First studies on traceless Staudinger ligation
acetylated
bis(p-dimethylaminoethyl)-phosphinomethanethiol
(15), which we intend to probe in acetylation studies of a benzylaz-
ide derivative and a more complex azido peptide in aqueous sys-
tems. Previously, phosphine 6 was obtained by Raines et al. via
the secondary phosphine borane 14 that was further reacted with
formaldehyde to give the tertiary hydroxymethyl phosphine bora-
ne.17a The alcohol was then transformed into the corresponding
mesylate and subjected to substitution with potassium thioacetate
to yield acetylthioester 15, which can be hydrolysed to deliver the
free thiol 6. This three-step procedure can be shortened by direct
alkylation of the secondary phosphine borane 14 with commer-
cially available acetylthiomethyl bromide.
After the successful synthesis of phosphine borane 15, we
wanted to evaluate its ability to mediate an acetyl transfer by
traceless Staudinger ligation in different solvent systems, espe-
cially buffer solutions, in comparison with commercially available
acetylthiomethyl(diphenyl)phosphine borane 16 to demonstrate
its potential applicability in chemical peptide modifications.10 For
this purpose, we first wanted to probe the reactivity of 15 on a
small water-soluble molecule. Therefore, we synthesized benzyl
azide 17 bearing a short polyethylene glycol chain in the meta po-
sition to enhance its water solubility (Scheme 3, for synthesis of 17
see Scheme 5 in Supplementary data).

S. Sowa et al. / Bioorg. Med. Chem. 21 (2013) 3465–3472
3467
1) n-BuLi (1.0 eq),
Et2O, -40°C - rt, 2 h
2) ethylene epoxide (1.0 eq),
Et2O, -40°C - rt, 2 h
MsCl (1.4 eq),
O
S
NEt3 (1.49 eq)
OH
O
O
Br
CH2Cl2, 0°C - rt, 48 h
Br
96%
Br
Br
84%
11
9
10
Me2NH (4+2 eq),
THF, 45°C, 48 h
97%
1 .Mg (3.3eq),
O
P
I2 cat.,THF, reflux, 3 h
2. (EtO)2P(O)H (1.0 eq),
THF, 0°C - rt, 24 h
H
N
Br
67%
N
N
13
12
1. DIBAL-H (5.1 eq)
CH2Cl2, rt, 1 h
46%
2. BH3-SMe2 (3 eq)
CH2Cl2, rt, 24 h
1. NaH (1.0 eq), DMF,
0°C, 20 min
2. BrCH2SAc (1.2 eq), DMF,
0°C - rt,14 h
BH3
BH3
P
S
P
H
O
60%
N
N
BH3
BH3
BH3
BH3
N
N
15
14
Scheme 2. Synthesis of phosphinothioester 15.
N3
O
BH3
P
O
6
S
O
NH
NH2
O
TFA
17 (1 eq)
+
R
rt, 1 h
40°C, 20 h
O
6
O
6
O
O
(1 eq)
R
19
18
15 R = -(CH2)2N(BH3)Me2
16
R = H
Scheme 3. Traceless Staudinger ligation with benzyl azide 17 after acidic borane deprotection.
In order to activate water-soluble 15 for traceless Staudinger
reactions, the borane protecting groups have to be removed first.
In principle, both acidic and basic deprotection strategies can be
envisioned, in which the latter one commonly proceeds best with
4 equiv DABCO in organic solvents like toluene or DMSO at
40 °C.11,12,14–16 We decided to apply acidic deprotection conditions
(e.g., TFA) for their better compatibility with the planned use of
aqueous solvent systems. As shown before by our group, the acidic
deprotection strategy has several advantages:16b,21 the reaction is
fast, proceeds at room temperature and TFA and borane are easily
removable by evaporation under vacuum for subsequent solvent
exchange to water. In addition, the acidic conditions do not yield
the oxophilic phosphine that undergoes the traceless Staudinger
ligation, but rather its protonated phosphonium salt. Thus, the sta-
bility towards air is enhanced and the reaction can be initiated by
addition of base and change of pH after addition of all reagents.22
During these studies it became evident that diphenylphosphi-
nomethanethiol 16 gave good results not only in organic solvents
(54% conversion in pure DMF, Table 1, entry 1), but also in mixed
aqueous solutions (73% conversion in 1:1 DMF/buffer, 0.4 M phos-
phate buffer, pH 8, Table 1, entry 7). Furthermore, in all transfor-
mations less than 10% P,N-bond hydrolysis to 19 were observed.
With further increase of the water content, the efficiency of the
ligation process with phosphine 16 dropped drastically. In con-
trast, the dimethylamine substituted derivative 15 reacted with
azide 17 to full conversion in a 1:4 mixture of DMF and buffer.

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S. Sowa et al. / Bioorg. Med. Chem. 21 (2013) 3465–3472
Table 1
Conditions for traceless Staudinger ligation of phosphines 15 and 16 with benzyl azide 17
Entry
Phosphine–borane
Solvent system
Ratioa (%)
17
18
19
1
2
3
4
5
6
7
8
16
15
16
15
16
15
16
15
DMF + DIPEA (12 equiv)
DMF + DIPEA (12 equiv)
Phosphate buffer (0.4 M, pH 8)
Phosphate buffer (0.4 M, pH 8)
Phosphate buffer (0.4 M, pH 8)/DMF (v:v 4:1)
Phosphate buffer (0.4 M, pH 8)/DMF (v:v 4:1)
Phosphate buffer (0.4 M, pH 8)/DMF (v:v 1:1)
Phosphate buffer (0.4 M, pH 8)/DMF (v:v 1:1)
39
54
50
74
69
0
54
39
44
21
31
88
73
45
7
7
6
5
0
12
4
8
23
47
a
Determined by LC–UV at 280 nm (normalised to 100%). For more information, see the SI.
Besides minor formation of some amine 19 by P,N-bond hydrolysis,
the sole product detected was the desired acetamide (Scheme 3). It
is also important to note that increasing the amount of the buffer
in the solvent system caused partial phosphine oxidation while
adjusting the pH. Therefore, it became necessary to work under
complete oxygen exclusion.
is noteworthy that a two-fold concentration of peptide 20 (Table 2,
entry 11) did not result in any improvement in conversion and
amide-to-amine ratio. The amount of observed amine side-product
was even higher. In contrast and as expected, conversion could be
increased by addition of more phosphine. With 10 equiv of phos-
phine 15 the product was formed in 61% with 33% of the undesired
amine 22 (Table 2, entry 10).
2.3. Traceless Staudinger ligation on azido peptides
3. Conclusion
These positive results encouraged us to investigate the reactiv-
ity of our compounds with azido peptide with unprotected side
chains. We decided to probe the efficacy of both borane protected
phosphino thioesters 15 and 16 in the reaction with an Fmoc-pro-
In summary, we presented an improved synthetic protocol for
bis{4-[(N,N-dimethylaminoethyl)phenyl]}(acetylthiomethyl)phos-
phine tris(borane) (15). In addition, we compared this water soluble
phosphine borane with commercially available diphenyl(acetylthi-
omethyl) phosphine borane (16) in selective acetylation reactions
via traceless Staudinger ligation with two different azido com-
pounds, both a water-soluble benzylic azide 17 and a more complex
tected peptide 20 containing e-azido-Lys in its structure (Scheme 4,
Table 2). The N-terminal Fmoc group was kept for better UV detec-
tion and comparison of the peptidic compounds by LC–MS. To our
knowledge, side chain acetylation of an azido norleucine peptide
via the traceless Staudinger ligation has not been presented in
the literature.
e-azido norleucine containing peptide 20. Compound 16 showed
superior reactivity under oxygen-free reaction conditions in the
acetyl transfer reaction with benzyl azide derivative 17 in a mixture
of buffer and DMF (1:1); but with increasing amounts of buffer (80%)
only phosphine 15 could yield an even higher product formation. For
peptide 20, only water soluble phosphine borane 15 was able to
mediate the traceless Staudinger ligation for selective lysine side
chain acetylation. The presented results show that water soluble
phosphine boranes could have broad utility as a ligation reagent
for selective peptide transformations as long as an oxygen-free envi-
ronment can be ensured.
The peptide was synthesized manually by SPPS following the
standard Fmoc protocol. All applied conditions for the traceless
Staudinger ligation with diphenylphosphine reagent 16 failed. In
every case we observed only traces of product 21 or no reaction oc-
curred at all. Due to the peptide’s hydrophobicity and therefore
poor solubility in aqueous systems, conversions in buffer were also
very slow for phosphine 15. Upon addition of DMF, the azido pep-
tide 20 started to dissolve and the reaction with phosphine 15 pro-
ceeded faster. The best conversion could be detected after 20 h in a
4:1 mixture of DMF and buffer. With only 3 equiv of phosphine we
observed 42% (Table 2, entry 6) of the desired acetylated peptide.
We can deduce from the amount of undesired amine 22 formed
during the reaction (29%, entry 6) that rearrangement of the aza-
ylide seems slower in the case of the peptide. Protonation of the
nitrogen might proceed faster, leading to the hydrolysis of the
aza-ylide. This could be due to the higher steric demand of the em-
ployed azido peptide 20 and the bulky Fmoc-group in close proxim-
ity. More test reactions with peptides not bearing the Fmoc group
on the N-terminus might be necessary to get more information. It
4. Experimental section
4.1. Materials and methods
4.1.1. Solvents and chemicals
All reactions were performed under argon atmosphere with dry
and degassed solvents. All reagents were purchased from commer-
cial suppliers and used without further purification. MeCN, DMF
were dried over molecular sieves or purchased dry from ACROS.
O
BH3
H
N
H
N
S
Fmoc-NH-Nle(N3)AESG-OH
20 (1 eq)
AESG
O
Fmoc
KAESG OH
P
OH +
Fmoc
O
TFA
R
rt, 1 h
40°C, 20 h
21
22
(3 eq)
R
HN
15 R = -(CH2)2N(BH3)Me2
16 R = H
Scheme 4. Traceless Staudinger ligation with azido peptide 20.

S. Sowa et al. / Bioorg. Med. Chem. 21 (2013) 3465–3472
3469
Table 2
4.2. General synthesis
Conditions for traceless Staudinger ligation of phosphines 15 and 16 with Fmoc-
protected azidopeptide 20
4.2.1. Synthesis of 2-(4-bromophenyl)ethanol 10
Entry Phosphine–
c (peptide) Solvent system
Ratioa (%)
The solution of 1,4-dibromobenzene (40 g, 0.17 mol) in dry
diethyl ether (200 mL) was cooled to À40 °C and n-buthyllithium
(105.6 mL, 1.6 M in hexanes, 0.17 mol) was added dropwise for
1 h. The mixture was allowed to warm up to rt and stirred for
2 h. The reaction was once again cooled to À40 °C and a solution
of ethylene epoxide (8.46 mL, 0.17 mol) in 40 mL of diethyl ether
was added slowly to the mixture. The reaction mixture was slowly
warmed to room temperature and stirred for 1 h at room temper-
ature. Then 40 mL of NH4Cl was added to quench the reaction. The
product was extracted with diethyl ether (3 Â 100 mL). The
organic layers were dried over MgSO4, filtered and concentrated.
Distillation afforded 28.63 g (85%) of a colourless liquid of 10. Bp
120–127 °C (1 mmHg). 1H NMR (400 MHz, CDCl3) d 1.60 (br s,
1H); 2.81 (t, J = 6.42 Hz, 2H); 3.83 (t, J = 6.60 Hz, 2H); 7.09–7.11
(m, 2H); 7.42–7.44 (m, 2H); 13C NMR (75 MHz, CDCl3) d 38.5;
63.3; 120.3; 130.7; 131.6; 137; GC tR = 6.406 min (s), 7 min (broad
peak); GC–MS (EI, 70 eV) m/z (%) 202 [M+] (24), 200 (25), 172 (22),
171 (71), 170 (24), 169 (71), 121 (15), 92 (9), 91 (100), 90 (63), 89
(53). For GC–MS results see SI (Figs. S1 and S2).
borane (equiv) (mM)
20 21 22
1
2
3
16 (3)
15 (3)
16 (3)
2
2
2
DMF + DIPEA (12 equiv)
DMF + DIPEA (12 equiv)
Phosphate buffer (0.4 M, pH
8)
Phosphate buffer (0.4 M, pH
8)
Phosphate buffer (0.4 M, pH
8)/DMF (v:v 1:4)
Phosphate buffer (0.4 M, pH
8)/DMF (v:v 1:4)
Phosphate buffer (0.4 M, pH
8)/DMF (v:v 9:1)
Phosphate buffer (0.4 M, pH
8)/DMF (v:v 1:1)
Phosphate buffer (0.4 M, pH
8)/DMF (v:v 1:4)
Phosphate buffer (0.4 M, pH
8)/DMF (v:v 1:4)
100
86 12
80 17
0
0
2
3
4
5
15 (3)
16 (3)
15 (3)
16 (3)
15 (3)
15 (5)
15 (10)
15 (3)
2
2
2
2
2
2
2
4
98
91
2
7
0
1
6
28 42 29
7
95
5
0
1
8
88 11
9
7 44 49
6 61 33
10
11
Phosphate buffer (0.4 M, pH
8)/DMF (v:v 1:4)
39 27 33
a
Determined by LC–UV at 301 nm (normalised to 100%). For more information,
4.2.2. Synthesis of 4-bromophenethyl methanesulfonate 11
To a solution of 10 (28.63 g, 0.142 mol) in 300 mL of CH2Cl2 was
added triethylamine (29.5 mL, 0.21 mol), the mixture was cooled
to 0 °C and mesyl chloride (15.4 mL, 0.2 mol) was added dropwise.
The reaction was stirred for 48 h at room temperature. Then 50 mL
of a 0.1 N HCl solution was added and the mixture was extracted
with CH2Cl2 (3 Â 150 mL). The organic layer was washed with
brine, dried over MgSO4, filtered and concentrated. The crude solid
was crystallized from a mixture of CH2Cl2 and hexane. The pure
compound 11 was obtained as a pale white solid (38.3 g, 96%).
Mp 58–60 °C. 1H NMR (400 MHz, CDCl3) d 2.90 (s, 3H); 3.02 (t,
J = 6.78 Hz, 2H); 4.40 (t, J = 6.97 Hz, 2H); 7.11–7.14 (m, 2H);
7.45–7.47 (m, 2H); 13C NMR (75 MHz, CDCl3) d 35.0; 37.4; 63.6;
121.0; 130.7; 131.8; 137.3; GC tR = 15.2 min; GC–MS (EI, 70 eV)
m/z 280 [M+] (0.1), 185 (11), 184 (98), 183 (12), 182 (100),
171(37), 169 (37), 104 (24), 103 (38), 91 (9), 90 (42), 89 (34); Anal.
Calcd for C9H11BrO3S: C, 38.72; H, 3.97. Found: C, 38.9; H, 3.88. For
GC–MS results see SI (Figs. S3 and S4).
see the SI.
THF, diethyl ether were dried over sodium/benzophenone ketyl
and DCM was dried over P2O5. The resins as well as Fmoc-pro-
tected natural Lamino acids were purchased from Novabiochem.
Thin-layer chromatography (TLC) was performed with precoated
silica gel plates and visualized by UV light (k = 254 nm), KMnO4
solution or iodine on silica gel. The reaction mixtures were purified
by column chromatography over silica gel (60–240 mesh).
4.1.2. Analytical methods
The NMR spectra were recorded on 500, 400 or 300 MHz spectrom-
eters in CDCl3 as a solvent at room temperature. Chemical shifts (d) are
reported in ppm relative to residual solvent peak (CDCl3; 7.26 ppm for
1H NMR and 77 ppm for 13C NMR). Mass spectra were recorded on
Shimazu GC–MS QP2010S spectrometer working in electron ionization
(EI) mode using a Phenomenex Zebron ZB-35HT INFERNO column with
the following parameters: pressure, 65 kPa; total flow, 33.9 mL/min;
column flow, 1.0 mL/min; linear velocity, 36.8 cm/s; split 30; 35 min
or 65 kPa; total flow, 23.9 mL/min; column flow, 1.0 mL/min; linear
velocity, 36.8 cm/s; split 20; 15 min in presence of internal standard
hexadecane. The analytical HPLC was applied from WatersTM with a
717 plus autosampler, a 600S controller, 2 pumps 616 and a 2489
UV/Visible detector connected to a 3100 mass detector. The RP-
4.2.3. Synthesis of 2-(4-bromophenyl)-N,N-dimethylethylamine
12
To a solution of mesylate 11 (38.3 g, 0.138 mol) in 200 mL of dry
THF dimethylamine (274 mL, 2 M in THF, 0.549 mol) was added. The
mixture was stirred at 45 °C for 24 h and then allowed to reach room
temperature. Dimethylamine (137 mL, 2 M in THF, 0.275 mol) was
once again added and the resulting solution was heated for another
day at 45 °C. The precipitate was filtered off and the filtrate was con-
centrated. The resulting residue was purified by flash column chro-
matography (chloroform/methanol, 15:1) to give 12 as an orange oil
28.77 g (97%). Rf = 0.6 (chloroform/methanol, 15:1). 1H NMR
(400 MHz, CDCl3) d 2.29 (s, 6H); 2.50–2.52 (m, 2H); 2.71–2.75 (m,
2H); 7.07–7.09 (m, 2H); 7.39–7.41 (m, 2H); 13C NMR (75 MHz,
HPLC-column was a Kromasil C18 (5
rate of 0.8 mL/min, MeCN/H2O (0.1% TFA)). Elementary analysis was
performed on PERKIN ELMER CHN 2400.
lm, 250 Â 4.6 mm with a flow
4.1.3. Preparative HPLC
HPLC purification of the peptide was performed on a JASCO LC-
2000 Plus system using a reversed phase C18 column (5 mL,
25 Â 250 mm, constant flow of 16.0 mL/min: 5 min at 7% MeCN
(with 0.1% TFA), gradient 7–95% MeCN (with 0.1% TFA) over
30 min (Gradient B)), consisting of a Smartline Manager 5000 with
interface module, two Smartline Pump 1000 HPLC pumps, a 6-
port-3-channel injection valve with 2.5 mL loop, a UV detector
(UV-2077) and a high pressure gradient mixer.
CDCl3)
d 33.7; 45.4; 61.2; 119.7; 130.3; 131.4; 139.3; GC
tR = 9.5 min; GC–MS (EI, 70 eV) m/z 228 [M+] (3), 226 (5), 171 (16),
169 (17), 104 (64), 103 (54), 102 (27), 91 (21), 90 (100), 89 (81).
For GC–MS results see SI (Figs. S5 and S6).
4.2.4. Synthesis of bis{4-[(N,N-dimethylamino)ethyl]phenyl}
(acetylthiomethyl)phosphine oxide 13
In a 500 mL flame-dried three-necked flask equipped with a re-
flux condenser magnesium turnings (1.88 g, 0.077 mol) were
placed under argon and 80 mL of anhydrous THF was added, fol-
lowed by 12 (15.05 g, 0.066 mol) and a catalytic amount of I2.
4.1.4. Peptide synthesis
Peptides were synthesised with ABI 433A Peptide Synthesizer
from Applied Biosystems via standard Fmoc-based conditions
(Fast-moc protocol with HOBt/HBTU conditions) on a preloaded
Wang resin (0.79 mmol/g).

3470
S. Sowa et al. / Bioorg. Med. Chem. 21 (2013) 3465–3472
The mixture was refluxed for 3 h until most of the magnesium was
consumed. The Grignard reagent partially precipitated. The result-
ing mixture was allowed to cool to room temperature and 50 mL of
anhydrous THF were added. The reaction mixture was cooled to
0 °C and diethyl phosphite (2.55 mL, 0.019 mol) was added. The
mixture was stirred for 24 h at room temperature. Then the reac-
tion mixture was cooled to 0 °C and was quenched with 10 mL of
brine and THF was evaporated. The residue was extracted with
CH2Cl2 (5 Â 150 mL), dried over MgSO4, filtered and concentrated.
The crude material was washed by flash column chromatography
(chloroform/methanol + 0.5% Et3N, 15:1) to elute less polar impuri-
ties and then the product was eluted with chloroform/methanol
(4:1 + 0.5% Et3N) to obtain a pale yellow thick oil (4.55 g, 67%).
Rf = 0.25 (chloroform/methanol 4:1). 1H NMR (400 MHz, CDCl3) d
2.30 (s, 12H); 2.55–2.57 (m, 4H); 2.81–2.85 (m, 4H); 7.31–7.4 (m,
4H); 7.57–7.63 (m, 4H); 8.04 (d, JP–H = 468.66 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 34.1 (d, JP–C = 1.2 Hz); 45.2; 60.7; 128.4 (d, JP–
C = 16.7 Hz); 129.2 (d, JP–C = 13.2 Hz); 129.5 (d, JP–C = 24.1 Hz);
130.7; 130.9; 145.3 (d, JP–C = 2.9 Hz); 31P (121 MHz, CDCl3) d
21.67 ppm (s).
(d, JP–C = 10.2 Hz), 132.8 (d, JP–C = 10 Hz), 142.3 (d, JP–C = 2.5 Hz),
193.1 (d, JP–C = 3.0 Hz). 31P NMR (202 MHz, CDCl3): d = 18.07 ppm
(m). HRMS (ESI-ToF): m/z = 445.2758 [MÀBH3+H]+ (calcd: m/
z = 445.2780) (Scheme 5).
4.2.7. Synthesis of tosylated hexaethylenglycol-
monomethylether 26
Hexaethyleneglycol monomethyl ether 24 (1 mL, 3.64 mmol)
was dissolved in dry acetonitrile (200 mL) and triethylamine
(1.01 mL, 7.29 mmol) was added. The mixture was cooled to 0 °C.
Solid p-toluenesulfonyl chloride (0.834 g, 4.37 mmol) was added
as well as trimethylamine hydrochloride (0.348 g, 3.64 mmol).
The resulting reaction mixture was stirred overnight at 0 °C. Then
the reaction mixture was allowed to warm to room temperature
and acetonitrile was evaporated. The residue was dissolved in
100 mL of CH2Cl2 and 50 mL of 1 M hydrochloric acid were added.
The mixture was extracted with CH2Cl2 (70 mL Â 5). The combined
organic fractions were dried over MgSO4 and the solvent was re-
moved under reduced pressure. The obtained product 26 was used
without any further purification (1.588 g, 96%). 1H NMR (500 MHz,
CDCl3) d 2.43 (s, 3H), 3.36 (s, 3H), 3.52–3.54 (m, 2H), 3.56 (br s, 3H),
3.58–3.61 (m, 5H), 3.62–3.64 (m, 10H), 3.65–3.68 (m, 2H), 4.13–
4.15 (m, 2H), 7.32–7.33 (m, 2H), 7.77–7.79 (m, 2H); 13C NMR
(125 MHz, CDCl3): d 21.6, 59.0, 68.6, 69.2, 70.4, 70.5, 70.5, 70.7,
71.9, 127.9, 129.8, 132.9, 144.7.
4.2.5. Synthesis of bis{4-[(N,N-dimethylamino)ethyl]phenyl}
phosphine tris(borane) 14
The solution of secondary phosphine oxide 13 (450 mg,
1.28 mmol) in anhydrous CH2Cl2 (5 mL) was added dropwise to
a solution of DIBAl-H (1.5 M in toluene, 4.26 mL, 6.4 mmol) un-
der argon in a 100 mL flame-dried three-neck flask. The resulting
solution was stirred for 1 h, then cooled to 0 °C. The solution
was diluted with anhydrous CH2Cl2 (20 mL) and argon was bub-
bled through the solution for 10 min. A solution of 2 N NaOH
(5 mL) was added dropwise to the reaction mixture, followed
by brine (5 mL). The resulting mixture was transferred to an
extraction funnel and the organic layer was separated, dried over
MgSO4, filtered under an inert atmosphere and concentrated un-
der reduced pressure to 10 mL. The resulting solution was cooled
to 0 °C with an ice bath under Ar and a borane dimethyl sulfide
complex (0.82 mL, 8.69 mmol) was added drop wise. The result-
ing reaction mixture was allowed to warm slowly to room tem-
perature overnight. The solvent was removed under reduced
pressure, and the crude oil was purified by flash column chro-
matography (silica gel, CH2Cl2) to give phosphine–borane com-
4.2.8. Synthesis of 3-(2-(2-(2-(2-(2-(2-methoxyethoxy)ethoxy)
ethoxy)ethoxy)ethoxy)ethoxy)benzyl alcohol 27
To a solution of tosylated hexaethylenglycol–monomethylether
26 (1.588 g, 3.55 mmol) and potassium carbonate (1.18 g,
8.53 mmol) in dry acetonitrile (40 mL), 3-hydroxybenzyl alcohol
25 (0.485 g, 3.9 mmol) was added. The resulting reaction mixture
was stirred for 48 h at reflux. Then, the reaction mixture was al-
lowed to reach room temperature and the solution was filtered
and concentrated. The residue was purified by column chromatog-
raphy (ethyl acetate/methanol, 10:1) to obtain a yellowish oil
(0.936 g, 79%). 1H NMR (500 MHz, CDCl3) d 3.36 (s, 3H), 3.52–
O
OH
plex 14 as
a
white solid in 49% yield (0.24 g). 1H NMR
6
24
(500 MHz, CDCl3) d 0.79–1.95 (br m, 9H), 2.66 (s, 12H), 2.93–
2.95 (m, 4H), 3.10–3.13 (m, 4H), 6.27 (dq, JP–H = 374 Hz, 1H),
7.30–7.33 (m, 4H), 7.60–7.62 (m, 4H); 13C NMR (125 MHz,
CDCl3) d 30.9, 51.9; 65.7; 127.0, 129.6 (d, JP–C = 9.99 Hz); 133.4
(d, JP–C = 10.0 Hz); 142.2 (d, JP–C = 2.7 Hz); 31P NMR (202 MHz,
CDCl3) d À0.12 ppm (m). Anal. Calcd for C20H38B3N2P: C, 70.18;
H, 9.42; N, 8.18. Found: C, 70.1; H, 9.23; N, 7.97.
TsCl (1.2 eq),
NEt3 (2.0 eq),
96%
Me3N*HCl (1.0 eq),
MeCN, 0 °C, 20 h
K2CO3 (2.4 eq),
MeCN, reflux,
OH
OH
O
48 h
O
+
OTs
6
4.2.6. Synthesis of bis{4-[(N,N-dimethylamino)ethyl]phenyl}
(acetylthiomethyl)phosphine tris(borane) 15
79%
OH
25
O
6
Secondary phosphine–borane 14 (200 mg, 0.54 mmol) was dis-
solved in dry DMF (2 mL) under argon and cooled to 0 °C. NaH
(22 mg, 0.54 mmol) was added and the mixture was stirred at
0 °C until bubbling stopped. Acetylthiomethyl bromide (110 mg,
0.65 mmol) was added and the resulting mixture was allowed to
warm to room temperature and stirred for 14 h. The solvent was
removed under reduced pressure and the residue was purified by
flash column chromatography (2% v/v ethyl acetate and 28% hex-
anes in CH2Cl2) to give phosphine–borane 15 as a colourless oil
in 60% yield. 1H NMR (500 MHz, CDCl3) d 2.26 (s, 3H), 2.66 (s,
12H), 2.94–2.96 (m, 4H), 3.10–3.13 (m, 4H), 3.76 (d, JP–
H = 6.94 Hz, 2H), 7.29–7.31 (m, 4H), 7.60–7.64 (m, 4H); 13C NMR
(125 MHz, CDCl3) d 23.5, 23.8, 30.1, 31.0, 51.9, 65.4 (d, JP–
C = 34.0 Hz), 125.8 (d, JP–C = 56.8 Hz), 126.9, 127.6, 128.5, 129.3
26
27
PBr3 (1.1 eq),
Et2O, 0 °C-rt,
20 h
74%
NaN3 (4.0 eq)
DMF, 60 °C,
24 h
N3
Br
82%
O
O
O
O
6
6
17
28
Scheme 5. Synthesis of benzyl azide 17.

S. Sowa et al. / Bioorg. Med. Chem. 21 (2013) 3465–3472
3471
3.54 (m, 2H), 3.61–3.64 (m, 13H), 3.65–3.67 (m, 3H), 3.70–3.72 (m,
4.2.13. Procedure of application of 15 or 16 in traceless
2H), 3.83–3.85 (m, 2H), 4.12–4.14 (m, 2H), 4.64 (d, JH-H = 3.6 Hz,
2H), 6.81–6.84 (m, 1H), 6.91–6.96 (m, 2H), 7.22–7.25 (m, 1H);
13C NMR (125 MHz, CDCl3) d 59.0, 65.1, 67.83, 69.7, 70.4, 70.5,
70.5, 70.6, 70.6, 70.8, 71.9, 113.0, 113.8, 119.2, 129.5, 142.7,
159.0; HRMS (ESI-ToF): m/z = 403.1943 [M+H]+ (calcd: m/
z = 403.2326).
Staudinger ligation with 17 in mixed solvent systems or pure
buffer (0.4 M phosphate buffer, pH 8)
Borane-protected phosphine 15 or 16 (3
according to the general procedure. To the residual phosphonium
salt of 15 or 16 were added 50 L solvent (Table 1, entries 3–8)
and azide 17 (1.283 mg, 3 mol) under argon. The pH was adjusted
lmol) was deprotected
l
l
to 8.0. The mixture was placed in a shaker and heated at 40 °C for
24 h. The ratio of azide 17, actamide 18 and amine 19 was deter-
mined by LC–UV (0% MeCN (0.1% TFA) for 5 min, 0–100% MeCN
(0.1%TFA) in 35 min, rt: 17: 28.010 min, 18: 21.542 min, 19:
18.730 min). The different molecule peaks were assigned by LRMS
(ESI-ToF): m/z (azide 17) = 450.2 [M+Na]+ (calcd: m/z = 450.2), m/z
(acetamide 18) = 444.0 [M+H]+ (calcd: m/z = 444.3), m/z (amine
19) = 402.2 [M+H]+ (calcd: m/z = 402.2). For LC–UV spectra see SI
(Figs. S9–S16).
4.2.9. Synthesis of 3-(2-(2-(2-(2-(2-(2-methoxyethoxy)ethoxy)
ethoxy)ethoxy)ethoxy)ethoxy)benzyl bromide 28
To a solution of PEG-functionalized benzyl alcohol 27 (0.6 g,
15 mmol) in dry diethyl ether (50 mL) phosphorus tribromide
(0.16 mL, 16.4 mmol) was added. The resulting reaction mixture
was stirred for 2 h at 0 °C. The mixture was allowed to warm to
room temperature and stirred overnight. Then the reaction mix-
ture was poured into 50 mL of ice water and extracted with diethyl
ether (50 mL Â 5). The combined organic fractions were dried over
MgSO4, filtered and the solvent was removed under reduced pres-
sure giving a yellow oil. Compound 28 was used without any fur-
ther purification (0.51 g, 74%). 1H NMR (500 MHz, CDCl3) d 3.37
(s, 3H), 3.53–3.55 (m, 2H), 3.62–3.66 (m, 14H), 3.67–3.68 (m,
2H), 3.71–3.73 (m, 2H), 3.84–3.86 (m, 2H), 4.12–4.14 (m, 2H),
4.45 (s, 2H), 6.83–6.85 (m, 1H), 6.94–6.97 (m, 2H), 7.21–7.24 (m,
1H); 13C NMR (125 MHz, CDCl3) d 59.0, 67.430, 69.5, 70.5, 70.5,
70.5, 70.6, 70.8, 71.9, 114.7, 115.2, 121.4, 129.7, 139.1, 158.9.
4.2.14. Procedure for synthesis of azido peptide 20
The peptide was synthesized manually by standard coupling
conditions following the Fmoc-protocol on a preloaded Fmoc-
Gly-Wang resin (0.79 mmol/g; 0.10 mmol)) The natural amino
acids (10 equiv) were coupled with HBTU, HOBt and DIPEA in
DMF for 1.5 h. N-Fmoc-6-azidonorleucine (2 equiv) was coupled
with HATU, HOBt and DIPEA in DMF for 6 h at rt. Purification by
preparative LCMS yielded the desired peptide 20 (27.90 mg,
38%): HRMS: m/z (ESI-ToF) [M+H]+ 739.3016, m/z (calcd) [M+H]+
739.3046.
4.2.10. Synthesis of 3-(2-(2-(2-(2-(2-(2-methoxyethoxy)ethoxy)
ethoxy)ethoxy)ethoxy)ethoxy)benzyl azide 17
4.2.15. Procedure of application of 15 or 16 in traceless
Staudinger ligation with 20 in DMF
Sodium azide (0.278 g, 4.3 mmol) was added to a solution of
benzyl bromide 28 in dry DMF (6 mL). The reaction was stirred
for 24 h at 60 °C. The reaction mixture was allowed to reach room
temperature and the solvent was evaporated. 20 mL of brine were
added to the resulting residue and the product extracted with
diethyl ether (5 Â 50 mL). The combined extracts were dried over
MgSO4, filtered and concentrated. Desired azide 17 was obtained
as a pale oil (0.374 g, 82%).
Borane-protected phosphine 15 or 16 (3
according to the general procedure. To a solution of the residual
phosphonium salt of 15 or 16 in DMF (250 L) under argon was
added azido peptide 20 (370 g, 0.5 mol) and DIPEA (1.1 L,
6.01 mol). The mixture was heated at 40 °C for 24 h and the ratio
lmol) was deprotected
l
l
l
l
l
of azide 20, actamide 21 and amine 22 was determined by LC–UV
(0% MeCN (0.1% TFA) for 5 min, 0–100% MeCN (0.1%TFA) in 35 min,
rt: 20: 24.726 min, 21: 22.436 min, 22: 21.463 min). The different
molecule peaks were assigned by LRMS (ESI-ToF): m/z (azide
20) = 739.2 [M+H]+ (calcd: m/z = 739.2), m/z (acetamide
21) = 755.2 [M+H]+ (calcd: m/z = 755.3), m/z (amine 22) = 713.2
[M+H]+ (calcd: m/z = 713.3). For LC–UV spectra see SI (Figs. S17
and S18).
1H NMR (400 MHz, CDCl3) d 3.37 (s, 3H), 3.52–3.55 (m, 2H),
3.62–3.66 (m, 14H), 3.67–3.68 (m, 2H), 3.71–3.73 (m, 2H), 3.84–
3.86 (m, 2H), 4.12–4.14 (m, 2H), 4.29 (s, 2H), 6.87–6.89 (m, 3H),
7.25–7.29 (m, 1H); 13C NMR (101 MHz, CDCl3) d 54.7, 59.0, 67.4,
69.6, 70.5, 70.5, 70.6, 70.6, 70.8, 71.9, 114.4, 114.4, 120.5, 129.8,
136.8, 159.1; HRMS (ESI-ToF): m/z = 428.2080 [M+H]+ (calcd: m/
z = 428.2391).
4.2.16. Procedure of application of 15 or 16 in traceless
Staudinger ligation with 20 in mixed solvent systems or pure
buffer (0.4 M phosphate buffer, pH 8)
4.2.11. General procedure of acidic deprotection of 15 or 16
Borane-protected phosphine 15 or 16 (3 lmol) was placed in an
eppendorf tube (0.5 mL) under argon and treated with 0.15 mL of
99% TFA. The mixture was placed in a shaker for 1 h at room tem-
perature. Then TFA was evaporated under high vacuum for 1 h.
Borane-protected phosphine 15 or 16 (3
3–8; 5 mol entry 9; 10 mol entry 10) was deprotected according
to the general procedure. To the residual phosphonium salt of 15 or
16 were added 250 L solvent (Table 2, entries 3–11) and azido
peptide 20 (370 g, 0.5 mol) under argon. The pH was adjusted
lmol Table 2, entries
l
l
l
4.2.12. Procedure of application of 15 or 16 in traceless
Staudinger ligation with 17 in DMF
l
l
to value 8. The mixture was placed in a shaker and heated at
40 °C for 24 h. The ratio of azide 20, actamide 21 and amine 22
was determined by LC–UV (0% MeCN (0.1% TFA) for 5 min, 0–
100% MeCN (0.1% TFA) in 35 min, rt: 20: 24.726 min, 21:
22.436 min, 22: 21.463 min). The different molecule peaks were
assigned by LRMS (ESI-ToF): m/z (azide 20) = 739.2 [M+H]+ (calcd:
m/z = 739.2), m/z (acetamide 21) = 755.2 [M+H]+ (calcd: m/
z = 755.3), m/z (amine 22) = 713.2 [M+H]+ (calcd: m/z = 713.3).
For LC–UV spectra see SI (Figs. S19–S26).
Borane-protected phosphine 15 or 16 (3
according to the general procedure. To a solution of the residual
phosphonium salt of 15 or 16 in DMF (50 L) was added azide
17 (1.283 mg, 3 mol), DIPEA (6.29 L, 36 mol). The mixture
lmol) was deprotected
l
l
l
l
was heated at 40 °C for 24 h and then the ratio of azide 17, acetam-
ide 18 and amine 19 was determined by LC–UV (0% MeCN (0.1%
TFA) for 5 min, 0–100% MeCN (0.1%TFA) in 35 min, rt: 17:
28.010 min, 18: 21.542 min, 19: 18.730 min). The different mole-
cule peaks were assigned by LRMS (ESI-ToF): m/z (azide
17) = 450.2 [M+Na]+ (calcd: m/z = 450.2), m/z (acetamide
18) = 444.0 [M+H]+ (calcd: m/z = 444.3), m/z (amine 19) = 402.2
[M+H]+ (calcd: m/z = 402.2). For LC–UV spectra see SI (Figs. S7
and S8).
4.2.17. Application of 15 in traceless Staudinger ligation with 20
at 4 mM concentration of azidopeptide 20 (Table 2, entry 11)
Borane-protected phosphine 15 or 16 (3
lmol) was deprotected
according to the general procedure. To the residual phosphonium

3472
S. Sowa et al. / Bioorg. Med. Chem. 21 (2013) 3465–3472
5. Robinson, P. J.; An, W.; Routh, A.; Martino, F.; Chapman, L.; Roeder, R. G.;
salt of 15 or 16 were added 250
buffer (4:1) and azido peptide 20 (738
l
L of a mixture of DMF and the
Rhodes, D. J. Mol. Biol. 2008, 381, 816.
l
g, 1.0 mol) under argon.
l
6. (a) Neumann, H.; Peak-Chew, S. Y.; Chin, J. W. Nat. Chem. Biol. 2008, 4, 232; (b)
Neumann, H.; Hancock, S. M.; Buning, R.; Routh, A.; Chapman, L.; Somers, J.;
Owen-Hughes, T.; van Noort, J.; Rhodes, D.; Chin, J. W. Mol. Cell 2009, 36, 153.
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14. Meyer, J.; Staudinger, H. Helv. Chim. Acta 1919, 2, 635.
The pH was adjusted to value 8. The mixture was placed in a shaker
and heated at 40 °C for 24 h. The ratio of azide 20, actamide 21 and
amine 22 was determined by LC–UV (0% MeCN (0.1% TFA) for
5 min, 0–100% MeCN (0.1% TFA) in 35 min, rt: 20: 24.726 min,
21: 22.436 min, 22: 21.463 min). The different molecule peaks
were assigned by LRMS (ESI-ToF): m/z (azide 20) = 739.2 [M+H]+
(calcd: m/z = 739.2), m/z (acetamide 21) = 755.2 [M+H]+ (calcd:
m/z = 755.3), m/z (amine 22) = 713.2 [M+H]+ (calcd: m/z = 713.3).
For LC–UV spectra see SI (Fig. S27).
Acknowledgments
The authors acknowledge financial support from the Ministry of
Higher Education, Poland (Grant No. N204 111 035), the German
Science Foundation within the Emmy-Noether program (HA
4468/2–1), the SPP 1623, the Einstein Foundation Berlin (Leibniz-
Humboldt Professorship), the SFB 765, the Boehringer-Ingelheim
Foundation (Plus 3 award), the Fonds der chemischen Industrie
(FCI) and the Studienstiftung des deutschen Volkes.
Supplementary data
15. Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006, 128, 8820.
16. For example: (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001, 3, 9;
(b) Kleineweischede, R.; Hackenberger, C. P. R. Angew. Chem., Int. Ed. 2008, 47,
5984; (c) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Org. Chem. 2002, 67, 4993;
(d) Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc.
2003, 125, 5268; Kalia, J.; Abbott, N. L.; Raines, R. T. Bioconjugate Chem. 2007,
18, 1064.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.045.
References and notes
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20. Unfortunately, we were not able to reproduce the results for the DIBAL-H
reduction which leads to a drastic drop in our overall yield.
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22. In addition, the acidic deprotection strategy allows also the global deprotection
of phosphino thioester peptides that can be applied in chemoselective peptide
cyclizations as shown by our group.16b
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