Acidic and basic deprotection strategies of borane-protected phosphinothioesters for the traceless Staudinger ligation

Article abstract of DOI:10.1016/j.bmc.2010.04.015

The traceless Staudinger ligation has recently found various applications in the field of peptide synthesis and modification, including immobilization and cyclization strategies. In this report, we utilize the traceless Staudinger ligation in the formation of amide bonds, which allows the acquisition of acylated aminosugars and peptides as well as the cyclization of peptides. A key element in these synthetic procedures is the use of a borane-protected phosphinomethanethiol, which is demonstrated to be prone towards oxidation in its unprotected form, during the synthesis of phosphinothioesters. In combination with acidic and basic deprotection strategies for the borane-protected phosphinothioesters, amide bonds can be formed in the presence of azides in moderate to good overall yields.

Full text of DOI:10.1016/j.bmc.2010.04.015

Bioorganic & Medicinal Chemistry 18 (2010) 3679–3686
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acidic and basic deprotection strategies of borane-protected
phosphinothioesters for the traceless Staudinger ligation
Michaela Mühlberg , Da’san M. M. Jaradat , Rolf Kleineweischede , Ilona Papp, Decha Dechtrirat,
*
Silvia Muth, Malgorzata Broncel, Christian P. R. Hackenberger
Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The traceless Staudinger ligation has recently found various applications in the field of peptide synthesis
and modification, including immobilization and cyclization strategies. In this report, we utilize the trace-
less Staudinger ligation in the formation of amide bonds, which allows the acquisition of acylated ami-
nosugars and peptides as well as the cyclization of peptides. A key element in these synthetic
procedures is the use of a borane-protected phosphinomethanethiol, which is demonstrated to be prone
towards oxidation in its unprotected form, during the synthesis of phosphinothioesters. In combination
with acidic and basic deprotection strategies for the borane-protected phosphinothioesters, amide bonds
can be formed in the presence of azides in moderate to good overall yields.
Received 18 January 2010
Revised 1 April 2010
Accepted 6 April 2010
Available online 9 April 2010
Keywords:
Traceless Staudinger ligation
Peptide ligation
Chemoselectivity
Azides
Ó 2010 Elsevier Ltd. All rights reserved.
Phosphines
Thioester
Glycosyl amides
1. Introduction
to form a natural amide bond by a capture/rearrangement strategy
comprised of a reversible trans-thioesterification and a subsequent
In recent years, the importance of chemoselective ligation and
modification strategies for protein synthesis has increased immen-
sely.1 These relatively new and often semi-synthetic methods,
including enzymatic protein ligation and native chemical ligation
(NCL), enabled the synthesis or modification of biologically impor-
tant peptides and proteins that were impossible to be accessed
alone by chemical methods, namely solid-phase peptide synthesis
(SPPS), or biological expression.2 The acquisition of homogenous
protein material is particularly important to study protein–protein
interactions, which requires minimal manipulations within the
proteins’ primary structure3 or post-translational modification
with small molecules, such as sugars4 or fatty acids,5 to investigate
possible interactions in detail. Protein semi-synthesis is often
accomplished by synthesis of the non-natural peptide fragment
by SPPS followed by chemoselective ligation to a second recombi-
nant fragment, yielding the desired manipulated protein.1,6 To
date, the most common ligation strategy is the NCL,7 which uses
a C-terminal peptide thioester and an N-terminal cysteine peptide
S?N shift.8 Although, this ligation method is widely used, it is lim-
ited due to its reliance on cysteine, which is the second least com-
mon residue.
In 2000, the Staudinger ligation was introduced as a new
chemoselective ligation strategy that allows the formation of
amide bonds starting from an azide and a phosphine, in which
the latter is covalently linked to an ester group.9 The aza-ylide
intermediate rearranges intramolecularly to produce an amide
linkage and a phosphine oxide, in which the two products are still
covalently bound to each other. The ligation is bio-orthogonal,
which implies a high selectivity of the two reaction partners azide
and phosphine. Consequently, high reaction rates of the intramo-
lecular acylation step enable execution of the reaction among bio-
molecules in an aqueous medium and in vivo applications.10
In order to make the reaction applicable for peptide ligations, a
traceless version of the Staudinger ligation was developed
(Scheme 1A).11,12 The phosphine is positioned in a thioester 1,
which first forms an intermediate iminophosphorane 3 by reaction
with an azide 2.13 An S?N shift leads then to the formation of the
amide 4 and liberation of the oxidized phosphine linker. Raines and
co-workers have developed several phosphine linkers to form dif-
ferent thioesters 1 for the traceless Staudinger ligation with azides
2.14 Since its discovery, the traceless Staudinger ligation has been
advanced to peptide couplings (Scheme 1A, R = amino acid or small
* Corresponding author. Fax: +49 (0)30 838 52551.
E-mail address: hackenbe@chemie.fu-berlin.de (C.P.R. Hackenberger).
These authors contributed equally to the work and should be considered as first
co-authors.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.015

3680
M. Mühlberg et al. / Bioorg. Med. Chem. 18 (2010) 3679–3686
I
R'
N3
O
O
O
R'
2
R'
N
II
(A)
(B)
R
S
PPh2
R
O
N
R
S
PPh2
- N2
H
4
1
3
oxidation
BH3
PPh2
O
BH3
O
O
NaOMe
R
X
Base
2
HS
O
PPh2
HS
4
S
PPh2
R
S
PPh2
MeOH
Δ
5
6
7a
1
BH3
R
X
Base
R
S
PPh2
Δ
7
in-situ
deprotection
2
4
BH3
PPh2
(C)
O
acid or base
= -(CH2)n-, peptide,
biaryl-scaffold
N3
S
N
H
8
O
Scheme 1. (A) Traceless Staudinger Ligation. (B) Phosphinothioester synthesis. (C) Acid or basic deprotection strategies for the cyclization of small molecules and peptides by
the traceless Staudinger ligation.
peptides)15 including
a
water-soluble version,16 and detailed
zation of unprotected peptides in the presence of several peptide
functionalities.23
mechanistic investigations,17 and various applications were per-
formed. Those studies included for instance the site-specific immo-
bilization of peptides on surfaces18 or the ligation of protected
(glyco-) peptide fragments.19 One of the most promising phosphine
linkers is the frequently applied diphenylphosphinomethanethiol
in 1. During the reaction pathway a favourable five-membered
transition state is formed in the final S?N shift to furnish the
amide bond. Thioesters 1 are usually synthesized by reaction of
activated carboxylic acids with free phosphinothiol 6, which can
be obtained by deacetylation of the commercially available acetyl
thioester 7a to borane-protected diphenylphosphinomethanethiol
5 and subsequent borane deprotection under basic conditions
(Scheme 1B).12,14,16,18a,19a,b
In conjunction with these investigations, we now present data
showing that the use of protected phosphinothiols in the synthesis
of (peptide) phosphinothioesters has a considerable practical
advantage, since the unprotected diphenylphosphinomethanethiol
6 is very sensitive towards oxidation. Consequently, we show that
these derivatives can be employed in a straightforward manner in
subsequent traceless Staudinger ligations in combination with
both deprotection strategies to peptide and carbohydrate
substrates.
2. Results and discussion
Alternatively but less frequently, 5 can be used directly in the
condensation to form a borane-protected thioester before borane
deprotection to 1 is initiated, again by basic treatment, which is
typically isolated before the traceless Staudinger ligation is in-
duced by azide addition.20,21
2.1. Stability studies of phosphinothiol linkers
Our first goal was to investigate the stability of the different
protected and unprotected phosphinothiol derivatives 5, 6 and
7a. In these studies, which were performed by 1H and 31P NMR
measurements over the course of 15 days, it was found that the
borane-protected acetyl thioester 7a as well as the de-acetylated
derivative 5 showed excellent stability upon air exposure, since
no oxidation products were determined within the detection limit.
In contrast, phosphinothiol 6 that was obtained by deprotection of
5 with DABCO in DMF over 4 h at 40 °C showed a fast oxidation
when left in an open flask. After 5 h about two third and after
15 h more than 90% of the phosphine 6 was oxidized already
(Scheme 1B and Fig. 1). As a result, although synthetically feasible
as demonstrated in previous publications,14–19 undesired oxidation
of 6 before or during thioester formation could limit the overall
yield especially for thioesters 1 when it is difficult to ensure strictly
inert conditions, for example, during protein conjugation in aque-
ous buffers or lysates.20
In addition to the intermolecular traceless Staudinger ligation,
also an intramolecular variant has been investigated. In this, the
borane-protecting group of the phosphine in phosphinothioesters,
which is known to prevent a Staudinger reaction with azides,22 is
utilized to install a phosphinothioester in an azidopeptide to en-
sure a traceless Staudinger ligation and thereby cyclization of bis-
functionalized azido-phosphinothioesters only upon deprotection
with bases (Scheme 1C).21 This protocol has been applied to the
synthesis of lactams as well as biaryl-containing natural product
analogues.21 Building on these investigations, our group has re-
cently developed a TFA deprotection strategy for medium-sized
azidopeptide phosphinothioesters that simultaneously releases
the borane and the peptide side chain protecting groups by TFA
addition and thereby allows a chemoselective intramolecular cycli-

M. Mühlberg et al. / Bioorg. Med. Chem. 18 (2010) 3679–3686
3681
In addition, the phosphorylated azidopeptide 13, which con-
tains residues 433–437 of the intrinsically unstructured Tau-pro-
tein,26 was chosen as another peptide substrate for an acetylation
by the traceless Staudinger ligation to compare the acidic and basic
deprotection strategies (Scheme 3). Since initial studies indicated a
slower conversion of azidopeptides when compared to azidogly-
cine 9, 10 equiv of 7a were used to counteract potential oxidation.
As described before, 7a was first deprotected and then directly em-
ployed for the traceless Staudinger ligation with 13. Basic depro-
tection with DABCO was performed in situ in presence of 13 and
both reactions were performed in DMF at 40 °C. After 16 h LC–
MS analysis revealed that only under the acidic deprotection con-
ditions a full conversion to 14 was observed, whereas under basic
conditions the reaction was finished after 40 h. To further enhance
the reaction rate, the basic route was repeated at 70 °C to acceler-
ate borane deprotection, as evident from the previous deprotection
study (see Table 1). Now full conversion of 13 was achieved after
16 h, however, the dehydroalanine peptide 15 was additionally ob-
served as by-product. These results imply that the traceless Stau-
dinger ligation with 13 proceeds slower under the basic
deprotection conditions and moreover, elevated temperatures lead
to elimination to the dehydroalanine. Therefore, the basic depro-
tection conditions should be avoided for phosphorylated peptide,
which favors the acidic deprotection strategy for this substrate.
Figure 1. Oxidation of phosphinothiol 6 in air.
2.2. Basic and acidic deprotection strategies for azide
transformations by the traceless Staudinger ligation
Building upon these results, we intended to employ borane-pro-
tected thioester 7a to the formation of simple acetylated amino
acids by the traceless Staudinger ligation with azidoglycine 9 as
model substrate under in situ-deprotection conditions. First, we
screened the best conditions for borane deprotection of 7a under
basic conditions to minimize reaction temperature and the re-
quired amount of base, in particular to allow also a later applicabil-
ity in polypeptide ligations. Deprotection studies of 7a revealed
complete borane deprotection after 20 min at 70 °C or after 4 h
at 40 °C with 3 equiv DABCO (1,4-diazabicyclo[2.2.2]octane) in
DMSO to yield thioester 1a (see Section 4, Table 1). Lowering the
amount of base or the use of DIPEA (diisopropylethylamine) only
led to incomplete deprotection. Transfer of the deprotection condi-
tions at 40 °C to intermolecular model reactions for the base in-
duced traceless Staudinger ligation of 7a with 9 furnished the
corresponding N-acetylglycine 10 in 78% conversion after 12 h in
DMSO-d6 or in 84% conversion after 12 h in DMF-d7 (Scheme 2A),
which supported the use of DMF for further investigations.
Next, we probed the use of acidic deprotection conditions for
the removal of the borane-protecting group in analogous in situ
traceless Staudinger ligations.23 To exclude thioester cleavage, a
short exposure of 7a to TFA, usually between 1 and 2 h, turned
out to be essential, in which absence of water in the cleavage cock-
tail minimized oxidation of the free phosphine.24 Again, azidogly-
cine 9 was used as a substrate for acetylation, which proceeded
quantitatively to 10 starting from 1.1 equiv 7a in DMF at 40 °C
within 12 h. During these studies it became evident that an excess
of base is necessary to avoid the formation of amine by-products,
since acidic conditions enhance the electrophilicity of the interme-
diate iminophosphorane and thus lead to hydrolysis by Staudinger
reduction (data not shown). In order to further compare the two
different deprotection strategies, acetylated b-GlcNAc azide 11
was used in an analogous reaction with 7a to yield a glycosyl
amide 12 (Scheme 2B).25 TFA deprotection of 7a and subsequent
traceless Staudinger ligation gave 12 in 62% yield. Alternatively,
basic deprotection with DABCO furnished 12 in a comparable yield
of 60%.
2.3. Borane-protected phosphinothiols in the synthesis and
cyclization of peptide phosphinothioesters
In a subsequent investigation, we turned our attention to the
synthesis of a more challenging C-terminal peptide-phosphino-
thioester by using borane-protected phosphinothiol 5. Although,
the basic deprotection could theoretically be applied on a synthet-
ically accessible borane-protected peptide-phosphinothioester 16
to allow a subsequent Staudinger ligation, an additional acidic
treatment is required to release the acid-labile side chain protect-
ing groups of the peptide (Scheme 4A). In addition to this practical
reason, the removal of the side chain protecting groups prior to the
formation of an amide bond by the traceless Staudinger ligation
has several advantages: First, side chain protecting groups could
sterically hinder peptide ligation or cyclization; second, the solu-
bility in polar solvents is increased which might prevent aggrega-
tion and finally, a chemoselective peptide ligation or cyclization is
possible, which in the case of peptide cyclization could lead to pre-
organization of the peptide chain and thus enhance intramolecular
amide bond formation.
First, we investigated the use of the borane-protected phos-
phinothiol 5 in the synthesis of the peptide-phosphinothioester
17. A model peptide comprising of 7 amino acids (AspAspPheGl-
nAspPheGly) was synthesized on an acid-lable TGT resin, which
was cleaved from the resin with 0.5% TFA in CH2Cl2. The C-termi-
nally deprotected peptide was converted into the thioester 16 by
activation with DIC. TFA/TIS-treatment rendered the fully depro-
Table 1
Borane-deprotection under basic conditions for 7a and in situ formation of 10: Temperature measurements after 1 h unless stated otherwise (r = ratio of deprotection/protection
according to –SCH2P– chemical shifts; ‘—’ = no deprotection observed; ‘fd’ = full deprotection). See also Section 4.2.2
Entry
Temperature (r value)
25 °C (r)
40 °C (r)
55 °C (r)
65 °C (r)
70 °C (r)
75 °C (r)
1
2
3
4
5
6
7
8
9
Heating only; without base
1 equiv DIPEA
2 equvi DIPEA
—
—
—
—
—
—
—
—
—
0.17:1
0.09:1
0.25:1
0.31:1
0.17:1
0.43:1
1.03:1
20 min: 1.25:1
60 min: 8.44:1
1.88:1
0.22:1
0.91:1
1.45:1
3 equiv DIPEA
2 equiv DABCO
3 equiv DABCO
3 equiv DABCO
—
0.20:1
0.39:1
1.15:1
4.68:1
20 min: fd
1 h: 2.2:1
4 h: fd

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M. Mühlberg et al. / Bioorg. Med. Chem. 18 (2010) 3679–3686
O
O
BH3
a) DABCO
OH
OH
(A)
(B)
N3
N
40 oC
H
S
PPh2
O
O
10
7a
9
b) TFA
c) 11, DIPEA
OAc
O
d) 11, DABCO
40 °C, DMF
BH3
PPh2
H
40 °C, DMF
AcO
AcO
O
N
S
60%
12
NHAc
62%
O
7a
12
b) TFA
c) 9, DIPEA
40 °C, DMF
10 > 95%
Scheme 2. Model studies for the intermolecular Staudinger ligation by basic (A) and acidic (B) deprotection of protected phosphinothioesters. Reagents and conditions: (a)
DABCO (3 equiv), deuterated solvents, 40 °C; (b) 99% TFA, 1 h, then TFA removal under high vacuum; (c) 9 or 11, DIPEA, DMF, 40 °C; (d) 11, DABCO, DMF, 40 °C. For exact
conditions see Section 4. TFA = trifluoroacetic acid, DIPEA = diisopropylethylamine, DABCO = 1,4-diazabicyclo[2.2.2]octane, DMF = dimethylformamide, DMSO =
dimethylsulfoxide.
a) TFA/TIS
c) 13, DABCO,
b) 13, DIPEA
40 °C, DMF
40 °C, DMF
7a
14
14
14
d) 13, DABCO,
70 °C, DMF
15
O
P
O
O
P
OH
OH
HO
HO
O
O
O
N3
OH
OH
OH
GSASLA
GSASLA
GSA
N
LA
N
N
H
H
H
O
13
14
15
Scheme 3. Acidic and basic deprotection of borane-protected phosphinothioester for traceless Staudinger ligation with azido phospho-serine peptide 15. Reagents and
conditions: (a) 97.5% TFA (2.5% TIS), 1 h, then TFA removal under high vacuum; (b) 13, DIPEA, DMF, 40 °C, 16 h; (c) 13, DABCO, DMF, 40 °C, 40 h; (d) 13, DABCO, DMF, 40 °C,
16 h. For exact conditions see Section 4.
tected peptide-phosphinothioester 17 in an excellent conversion
with only minimal amounts of oxidized peptide as a side prod-
uct, thereby demonstrating the benefits of the acid deprotection
strategy for unprotected peptide-phosphinothioesters (see
Section 4).
aration delivered the Arg-containing cyclic peptide 19a in overall
11% yield over three steps, which included the phosphinothioester
synthesis, the global TFA deprotection and the traceless Stauding-
er cyclization. In contrast, the purification of the Lys-containing
cyclic peptide 19b, which was detected in the crude reaction mix-
ture by LC–MS analysis, turned out to be impossible, due to signif-
icant amount of the Lys-side chain cyclized product. The
formation of this unwanted cyclization product can be attributed
to the deprotonation of the Lys side chain with an excess of DIPEA
present, which attacks the labile thioester. Several attempts to
optimize this reaction by varying the equivalents of DIPEA used
to scavenge residual TFA led to no significant improvement for
the formation of 19b. Despite this limitation in the synthesis of
cyclic peptide containing Lys residues, we chose a larger head-
to-tail cyclic variant of the circular bacterial protein Microcin
J25 as the final synthetic target.27 This 21 amino acid comprising
polypeptide has previously been synthesized in a head-to-tail var-
iant by an intramolecular NCL followed by heterogeneous desul-
furization conditions, which delivered the cyclic peptide 19c in
25% yield over two steps starting from the peptide thioester.28
Our analogous strategy by the traceless Staudinger ligation
yielded head-to-tail cyclic 19c in 12% overall yield, which how-
Finally, we subsequently subjected this protocol to the synthe-
sis of cyclic peptides. Previous studies in our lab have demon-
strated that medium sized peptides can be yielded by such a
traceless Staudinger cyclization strategy in yields between 20%
and 36% over three synthetic steps.23 In these examples, we have
shown that the reaction proceeds in the presence of amino acid
side chain functional groups including a carboxylic acid, alcohol
and phenol. Here, we intended to probe the performance of
unprotected nucleophilic peptide side chains such as amines
and guanidines in Arg and Lys for interference with the S?N shift
during the traceless Staudinger ligation (Scheme 4B). Two azido-
peptide thioesters 18a and 18b were synthesized including either
an arginine or a lysine residue (see Scheme 4 for sequence).
Deprotection with TFA and cyclization in DMF were performed
by previously established conditions. In both cases side products
were detected which were identified as oxidized thioesters and
non-cyclized peptides with an N-terminal amine. Preparative sep-

M. Mühlberg et al. / Bioorg. Med. Chem. 18 (2010) 3679–3686
3683
PG
PG
PG
basic
deprotection
BH3
O
O
a) 0.5% TFA
O
DDFQDFG
DDFQDFG
16
S
PPh2
(A)
S
PPh2
DDFQDFG
b) 5, DIC,
DMAP
BocHN
BocHN
BocHN
O
c) TFA
TGT-resin
side-chain
deprotection
acidic
deprotection
O
DDFQDFG
17
S
PPh2
H2N
PG
PG
BH3
(B)
O
S
a) 0.5% TFA
O
O
N3
O
O
PPh2
Peptide
N3
Peptide
b) 5, DIC,
DMAP
N
N
H
H
18
O
c) TFA/TIS
HN Peptide
then
DIPEA
NH
19a
19b
19c
O
C
H2
19
Scheme 4. (A) Peptide-phosphinothioester synthesis by acid deprotection. (B) Cyclization of peptides by the traceless Staudinger ligation. Reagents and conditions: (a) 0.5%
TFA, 2.5% TIS, CH2Cl2, 2 h; (b) 5 (1.5 equiv), DIC (3 equiv), DMAP (cat.), CH2Cl2, 8 h; (c) 97.5% TFA, 2.5% TIS, 1 h, then precipitation from dry ethyl ether. For further information
see Section 4.
ever included the peptide-phosphinothioester synthesis in con-
trast to the previously mentioned study.
sents
reactions.
a promising strategy to other amide bond formation
3. Conclusion
4. Experimental section
In conclusion, the presented results support the use of the bor-
ane-protected diphenylphosphinomethanethiol (5) as an expedi-
ent phosphine linker for the traceless Staudinger ligation of small
molecules as well as carbohydrates and larger peptides. The borane
group preserves phosphine stability against oxidation and enables
triggering of the traceless Staudinger ligation with azides, which is
essential for cyclization strategies. So far, acidic and basic depro-
tection conditions have been established for borane deprotection
of phosphinothioesters. The basic deprotection strategy proceeds
well in organic solvents and gives good yields in subsequent reac-
tions. Nevertheless, the rapid oxidation of the diphenylphosphino-
methanethiol may limit the application of the traceless Staudinger
ligration in biological systems, since the evaluated deprotection
conditions are too harsh in this environment. The acidic deprotec-
tion with TFA can be performed at room temperature, thus deliver-
ing free phosphine and simultaneously removing side chain
protecting groups in peptides. Hence, the chemoselectivity of the
traceless Staudinger ligation can be addressed which broadens
the scope of this amide bond forming reaction. One potential draw-
back in the use of the acidic deprotection strategy is the final addi-
tion of bases, such as DIPEA, to scavenge residual TFA to avoid the
competing Staudinger reduction of the azide to an amine. This pro-
cedure may limit the chemoselectivity of the traceless Staudinger
ligation, since under these basic conditions Lys side chains can
form an amide bond with the phosphinothioester as demonstrated
in cyclization studies in small peptides. Nevertheless, the pre-
sented results show that the application of the borane-protected
phosphinomethanethiol in the traceless Staudinger ligation pre-
4.1. Materials and methods
4.1.1. Solvents and chemicals
All reactions were performed under argon unless stated other-
wise. Solvents such as ethyl acetate, hexane and CH2Cl2, were pur-
chased as p.a. grade and distilled once prior to use. Reagents—
including deuterated as well as dry solvents and acetonitrile—are
commercially available as reagent grade and did not require fur-
ther purification. The resins as well as Fmoc-protected natural L-
amino acids were purchased from Novabiochem. Flash column
and thin layer chromatography were performed with Merck Silica
Gel 60. TLC analysis was carried out under ultraviolet light
(k = 254 nm) or via additional staining with p-anisaldehyde. Azi-
do-glycine (9),29 2-Acetamido-2,4,6-tri-O-acetyl-2-deoxy-b-
D-
glucopyranosyl azide (11)30 and borane-protected diphenyl-
phosphinomethanethiol acetate (7a)31 were synthesized by
published protocols.
4.1.2. Analytical methods
An Agilent 6210 ToF LC/MS system (Agilent Technologies, Santa
Clara, CA, USA) with ESI source was used for mass detection of the
peptides. H2O and MeCN (both including 1% acetic acid) were used
as eluent. The flow rate was 0.5 ml/min. Conversion of peptide
reactions was determined by LC/MS analysis (C18-column, constant
flow of 0.5 ml/min: 3 min at 3% MeCN (with 1% AcOH), gradient 3–
100% MeCN (with 1% AcOH) over 19 min (Gradient A)). 1H, 13C and
31P NMR spectra were recorded on a Jeol ECX-400 400 MHz spec-
trometer at ambient temperature.

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M. Mühlberg et al. / Bioorg. Med. Chem. 18 (2010) 3679–3686
4.1.3. Preparative HPLC
HPLC purification of the peptides was performed on a JASCO LC-
2000 Plus system using a reversed phase C18 column (5 m,
25 Â 250 mm, constant flow of 18.9 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.
and the conversion to N-acetylglycine (10) was determined by 1H
NMR analysis after 12 h. The analytical data was in accordance
with the commercially available N-acetylglycine (Aldrich): 1H
NMR (400 MHz, DMF-d7): d = 8.58 (br s, 1H), 7.73–7.77 (m, 1H),
3.63–3.67 (m, 2H), 1.95 (s, 3H).
l
4.2.4. Synthesis of N-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
D-glucopyranosyl) acetamide (12)
Route 1 (acidic deprotection conditions): 20 mg (0.069 mmol)
phosphinothioester 7a was treated with 1 ml 99% TFA for 1 h and
the volatiles were removed under high vacuum. The residual phos-
phonium salt was dissolved without further purification in 1 ml
dry DMF and the solution was degassed by three cycles of vacuum
and argon. DIPEA (12 equiv) as well as 1 equiv of glycosyl azide 11
were added to this solution and the resulting reaction mixture was
stirred for 18 h at 40 °C before the reaction mixture was concen-
trated under high vacuum. The residue was chromatographed on
a silica gel column with chloroform–methanol (19:1). Pure acety-
lated GlcNAc (12) was obtained in 62% yield (16.7 mg,
0.043 mmol).
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
TGT resin (0.28 mmol/g).
4.2. General synthesis
4.2.1. Stability studies of diphenylphosphinomethanethiols 5, 6
and 7a
The borane-protected phosphine species 5 (1 equiv) was dis-
solved in dry DMF (1 mL) under argon. DABCO (3 equiv) was
added, and the mixture was heated at 40 °C for 4 h. Afterwards,
the solvent was removed under reduced pressure and the residue
was left in an open flask and monitored over a time period of 1–
24 h by 1H NMR and 31P NMR. Figure 1 depicts the oxidation of
phosphine 6 based on 1H NMR analysis after 1, 2, 5 and 20 h (the
integral ratio (phosphine oxide/phosphine) is shown): 31P NMR
(121 MHz, CDCl3): 26.7 (P@O), À7.5 (P).
Route 2 (basic deprotection conditions): 20 mg (0.069 mmol)
phosphinothiol 7a was dissolved in 1 ml dry DMF and the solution
was degassed by three cycles of vacuum and argon. 3 equiv of DAB-
CO as well as 1 equiv of glycosyl azide 11 were added. The temper-
ature was elevated to 40 °C and the reaction mixture was stirred
for 18 h. The residue was chromatographed on a silica gel column
with chloroform–methanol (19:1). acetylated GlcNAc (12) was ob-
tained in 60% yield (14 mg, 0.031 mmol): 1H NMR (500 MHz,
CDCl3): d = 7.09 (d, 1H), 6.07 (d, 1H), 5.23 (t, 1H), 5.10–5.25 (m,
2H), 4.39 (dd, 1H) 4.16–4.25 (m, 2H), 3.85 (m, 1H), 2.19, 2.17,
2.14, 2.08, 2.06 (5s, 15H, Ac); HRMS (ESI-ToF): m/z = 389.159
[M+H]+ (calcd: m/z = 389.156), 411.137 [M+Na]+ (calcd: m/z =
411.137). Further analytical analysis is in accordance with reported
results.32
Borane-protected phosphines 5 and 7a were left in an open
flask in air and monitored over a time period of 15 days by 1H
NMR and 31P NMR. 31P NMR (121 MHz, CDCl3): d = 19.5 (d, P-
BH3, 7a); 22.5 (d, P-BH3, 5).
4.2.2. Borane-deprotection under basic conditions for 7a and
in situ formation of 10
4.2.5. Synthesis of N-acetyl phospho-serine peptide 14
Peptide 13 was synthesized on a preloaded Wang-resin and azi-
do glycine 9 was coupled manually using 5 equiv amino acid, HOBt,
HBTU and DIPEA in DMF. The peptide was then purified by pre-
parative HPLC and analysed by HRMS (ESI-ToF): m/z = 611.223
[M+H]+ (calcd: m/z = 611.219).
The optimal basic deprotection conditions of 7a were carried
out with DIPEA and DABCO as bases. 7a was dissolved in an NMR
tube using DMSO-d6 deuterated solvent. Deprotection was moni-
tored out at different temperatures, starting from rt up to 75 °C,
and different equivalents of DIPEA were added (1, 2 and 3 equiv).
For comparative purposes, the same tests were then carried out
using DABCO as a base (see Table 1). The second model test was
carried out to optimize the basic deprotection conditions. Here,
thioester 7a was dissolved in DMSO-d6, to which the azido-glycine
9 and 3 equiv of DABCO were added and the reaction was carried
out at 40 °C. Again these tests were done in NMR tubes. Progress
was checked on an hourly basis until the desired product 10 was
obtained in the absence of the starting materials.
Subsequently, 7a (0.32 mmol) was dissolved in 0.7 ml deuter-
ated solvent (DMSO-d6 or DMF-d7) in an NMR tube to which
108 mg DABCO (0.96 mmol, 3 equiv) and 34 mg azidoglycine (9)
(0.32 mmol, 1 equiv) were added. The reaction was carried out at
40 °C and the conversion to N-acetylglycine (10) was determined
by 1H NMR analysis (see Scheme 3A). The analytical data was in
accordance with the commercially available N-acetylglycine (Al-
drich): 1H NMR (500 MHz, DMSO-d6/DMF-d7): d = 8.58 (br s, 1H),
7.73–7.77 (m, 1H), 3.48–3.52 (m, 2H), 1.81 (s, 3H).
Route 1 (acidic deprotection conditions): 9.5 mg (32.8 lmol,
10 equiv) Phosphinothioester 7a was treated with 1 ml 97.5% TFA
(2.5% TIS) for 1 h and the volatiles were removed under high vac-
uum. The residual phosphonium salt was dissolved without further
purification in 1 ml dry DMF and the solution was degassed by
three cycles of vacuum and argon. 5 equiv of DIPEA as well as
1 equiv of azido peptide 13 were added to this solution and the
resulting reaction mixture was stirred for 16 h at 40 °C. Conversion
was checked by LCMS (ESI-ToF): m/z = 627.242 [M+H]+ (calcd: m/
z = 627.239). The product was eluted at 12.8 min (constant flow:
5% CH3CN (with 1.0% AcOH), gradient: 5–65% CH3CN (with 1.0%
AcOH) over 25 min; 65–100% CH3CN (with 1.0% AcOH) over 1 min).
Route 2 (basic deprotection conditions): 9.5 mg (0.069 mmol)
phosphinothiol 7a was dissolved in 1 ml dry DMF. The solution
was degassed by three cycles of vacuum and argon. 3 equiv of DAB-
CO as well as 1 equiv of azido peptide 13 were added. The temper-
ature was elevated to 40 °C (a) or 70 °C (b), respectively.
Conversion was determined by LCMS after 16 and 40 h: HRMS
(ESI-TOF): (a) after 40 h: m/z = 627.242 [M+H]+ (calcd: m/
z = 627.239); (b) after 16 h: m/z = 627.245 [M+H]+ (calcd: m/
z = 627.239); m/z = 529.267 [MÀH3PO4+H]+ (calcd: m/z = 529.262).
4.2.3. Acidic deprotection of 7a and application in the traceless
Staudinger ligation with azidoglycine 9
Acetyl phosphinothioester 7a (0.176 mmol, 1.1 equiv) was trea-
ted with 0.5 ml 99% TFA. After stirring for 1 h the mixture was con-
centrated, dissolved in 0.35 ml DMF, 16.2 mg azidoglycine (9)
(0.160 mmol, 1 equiv) and 0.34 ml DIPEA (1.92 mmol, 12 equiv)
were added. The reaction was carried out at room temperature
4.2.6. Synthesis of peptide phosphinothioester 17
The peptide was synthesized on an ABI 433a peptide synthe-
sizer on a TGT-resin (Novabiochem) with the first amino acid

M. Mühlberg et al. / Bioorg. Med. Chem. 18 (2010) 3679–3686
3685
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(Gly) already attached to the resin. The last amino acid was intro-
duced with an N-terminal Boc-protection. The peptide was cleaved
from the resin with 0.5% TFA in CH2Cl2 (including 2.5% TIS) for 2 h.
The resin was filtered off and washed with CH2Cl2. The filtrate and
the washing solution were combined, and the solvent was re-
moved under high vacuum. The C-terminally deprotected peptide
was then dissolved in dry CH2Cl2 (5 ml, 0.033 mmol). After the
addition of 3 equiv DIC and catalytic amounts of DMAP, borane-
protected diphenylphosphinomethane-thiol 5 (5 equiv) was added
and the reaction mixture was stirred for 12 h. The crude reaction
mixture containing the protected phopshinothioester 16 was trea-
ted with a solution containing 97.5% TFA and 2.5% TIS (0.5 ml,
0.033 mmol) for 1 h. The globally deprotected peptide thioester 17
was precipitated from 10 ml dry ethyl ether and characterized by
LC–MS.
2. (a) Meister, S. M.; Kent, S. B. H. Peptides: Structure and Function; Pierce Chemical
Co.: Rockford, IL, 1983. 103; (b) Bird, G. H.; Lajmi, A. R.; Shin, J. A. Anal. Chem.
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Analytical HPLC and HRMS (ESI-TOF): m/z = 1057.3638 [M+H]+
(calcd: m/z = 1057.3525) peptide 17 eluted at 15.84 min (Gradient
A).
4.2.7. Peptide cyclization by the traceless Staudinger ligation
The azidopeptide was synthesized on an ABI 433a peptide syn-
thesizer on a TGT-resin (Novabiochem) with the first amino acid
(Gly) already attached to the resin. The peptide was cleaved from
the resin with 0.5% TFA in CH2Cl2 (including 2.5% TIS) for 2 h.
The resin was filtered off and washed with CH2Cl2. The filtrate
and the washing solution were combined, and the solvent was re-
moved under high vacuum. The C-terminally deprotected peptide
was dissolved in dry CH2Cl2 (5 ml, 0.033 mmol). After the addition
of 3 equiv DIC and catalytic amounts of DMAP, borane-protected
diphenylphosphinomethane-thiol 5 (1.5 equiv) was added and
the reaction mixture was stirred for 12 h. The crude reaction mix-
ture containing the protected phosphinothioester 18 was treated
with a solution containing 97.5% TFA and 2.5% TIS (0.5 ml,
0.033 mmol) for 1 h. The globally deprotected thioester was pre-
cipitated from 10 ml dry ethyl ether, characterized by LC–MS to
verify conversion to 18 and re-dissolved in dry DMF (5 ml,
0.033 mmol). For the cyclization 20 equiv DIPEA were added to
the reaction mixture and the reaction mixture was stirred for
12 h. The cyclized peptides 19 were purified by preparative HPLC
(Gradient B). For yields see Scheme 4B. LC/HRMS analysis was car-
ried out to confirm the identity of the final products and the pep-
tide intermediates.
5. Brunsveld, L.; Kuhlmann, J.; Alexandrov, K.; Wittinghofer, A.; Goody, R. S.;
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Danishefsky, S. J. J. Am. Chem. Soc. 2004, 126, 6576; (d) Hofmann, R. M.; Muir, T.
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Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001, 3, 9.
13. Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
14. Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Org. Chem. 2002, 67, 4993.
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16. (a) Tam, A.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc. 2007, 129, 11421; (b)
Tam, A.; Raines, R. T. Bioorg. Med. Chem. 2009, 17, 1055.
17. (a) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006, 128, 8820;
(b) Tam, A.; Soellner, M.; Raines, R. T. Org. Biomol. Chem. 2008, 6, 1173.
18. (a) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc.
2003, 125, 11790; (b) Köhn, M.; Wacker, R.; Peters, C.; Schroeder, H.; Soulere,
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Angew. Chem., Int. Ed. 2006, 45, 1408.
Analytical HPLC and HRMS (ESI-TOF): 19a: m/z = 1133.5745
[M+H]+ (calcd: m/z = 1133.5848); 19c: m/z = 2107.0337 [M+H]+
(calcd: m/z = 2107.0393), 1054.0136 [M+2H]2+ (calcd: m/z =
1054.0236). The peptide 19a eluted at 13.67 min and 19c eluted
at 16.09 min (Gradient A).
19. (a) Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc.
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20. For the use of borane-protected phosphinothioesters in the conjugation of
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5984.
Acknowledgements
The authors acknowledge financial support from the German
Science Foundation (DFG) within the Emmy-Noether program
(HA 4468/2-1), the Sonderforschungsbereich 765 ‘Multivalency’
and the Fonds der Chemischen Industrie (FCI, doctoral scholarship
to M.M.) The Royal Scientific Society of Jordan is acknowledged for
a Scholarship to D.M.M.J.
24. For small molecules the use of scavengers does not influence the overall yield,
however, addition of TIS is recommended when peptides are employed (see
also Ref.23).
25. For other glycosylamide syntheses using the traceless Staudinger ligation, see:
(a) He, Y.; Hinklin, R. J.; Chang, J.; Kiessling, L. L. Org. Lett. 2004, 6, 4479; (b)
Bianchi, A.; Bernardi, A. Tetrahedron Lett. 2004, 45, 2231; (c) Bianchi, A.; Russo,
A.; Bernardi, A. Tetrahedron: Asymmetry 2005, 16, 381; (d) Bianchi, A.; Bernardi,
A. J. Org. Chem. 2006, 71, 4565; (e) Schierholt, A.; Shaikh, H. A.; Schmidt-Lassen,
J.; Lindhorst, T. K. Eur. J. Org. Chem. 2009, 3783.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.015.
26. Goedert, M.; Spillantini, M. G.; Jakes, R.; Rutherford, D.; Crowther, R. A. Neuron
1989, 3, 519.
References and notes
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