Chemoselective peptide cyclization by traceless staudinger ligation

Article abstract of DOI:10.1002/anie.200801514

(Chemical Equation Presented) Two birds with one stone! An intramolecular traceless Staudinger ligation was employed to synthesize cyclic peptides. The final amide bond formation was induced by deprotection of the azidopeptide phosphinothioesters. Treatment with trifluoroacetic acid (TFA) results in the simultaneous removal of the borane along with the protecting groups on the peptide side chains, and chemoselective amide bond cyclization ensues.

Full text of DOI:10.1002/anie.200801514

Communications
DOI: 10.1002/anie.200801514
Staudinger Ligation
Chemoselective Peptide Cyclization by Traceless Staudinger
Ligation**
Rolf Kleineweischede and Christian P. R. Hackenberger*
Dedicated to Professor Michael Bienert on the occasion of his 65th birthday
Complex cellular function is controlled by a network of
protein–protein interactions which are often individually
regulated by the post- or cotranslational attachment of
specific functionalities to protein side chains, a process
commonly referred to as protein modification.[1,2] To probe
the specific impact of a particular modification on the
proteinꢀs biological function, efforts have been focused on
the acquisition of homogeneous protein material containing
natural protein modifications, including glycosylation and
phosphorylation, for comparative studies with their unmodi-
fied analogues.[2] The most effective semisynthetic strategy in
protein synthesis employs the expressed protein ligation
(EPL).[2a] In this chemoselective process an unprotected
synthetic peptide fragment containing the particular modifi-
cation of interest is ligated with an unmodified expressed
protein by native chemical ligation (NCL) at a Cys junction.[3]
In contrast to impressive studies in which the biological
and structural function of various protein modifications have
been validated by this approach,
tion event on the structural stability and biological activity.[6]
Common synthetic strategies towards cyclic polypeptides
either employ the cyclization of a peptide with protected side
chains using activation reagents in solution or on the solid
support,[7] or an intramolecular chemoselective amide bond
formation by NCL.[8] Whereas the latter approach can directly
deliver unprotected Cys-containing cyclic polypeptides and
thereby utilize the advantages of structure-assisted intra-
molecular ring closure,[9] non-Cys-containing peptides have
been generated by additional chemical transformations on the
cyclization product including Cys desulfurization[10] and
auxiliary-based methodologies.[11]
To develop a direct synthetic strategy for non-Cys-
containing head-to-tail cyclic polypeptides, which would
allow access to important members of the posttranslationally
modified cyclic proteins like the 70-membered Bacteriocin
AS48,[12] we decided to employ the traceless Staudinger
ligation[13] for the conjunction of the peptide termini in small
only
a few investigations have
addressed the functional conse-
quence of protein cyclization.[4] The
protein head-to-tail cyclization rep-
resents an interesting posttransla-
tional modification process, which
occurs almost exclusively on riboso-
mally expressed proteins in bacterial
organisms and less frequently in
mammals and plants.[5] Most impor-
tantly, cyclic proteins display prom-
ising biological activity (especially
antibacterial) and remarkable stabil-
ity, thus making them ideal candi-
dates for detailed investigations con-
cerning the influence of the cycliza-
Scheme 1. A) Traceless Staudinger ligation.[14] B) Base-induced Staudinger cyclization.[22]
DABCO=1,4-diazabicyclo[2.2.2]octane.
peptide model systems. The traceless Staudinger ligation,
which was first described by the groups of Raines and
Bertozzi,[14a,b] originates from the Staudinger reaction[15] of an
azide with a phosphine yielding an iminophosphorane, which
reacts intramolecularly as the aza ylide with an internal
electrophile, in this case a thioester, to yield a native amide
bond (Scheme 1A). Consequently, the traceless Staudinger
ligation, in contrast to NCL, does not require a Cys residue at
the ligation site. Since its discovery in 2000, the traceless
Staudinger ligation has been at the focus of extensive
methodology studies,[14] including a recent description of a
water-soluble variant,[16] detailed mechanistic investiga-
[*] R. Kleineweischede, Dr. C. P. R. Hackenberger
Institut für Chemie und Biochemie
Freie Universität Berlin
Takustrasse 3, 14195 Berlin (Germany)
Fax: (+49)30-838-52551
E-mail: hackenbe@chemie.fu-berlin.de
[**] The authors acknowledge financial support from the German
Science Foundation (DFG) within the Emmy-Noether program
(HA 4468/2-1) and the SFB765 “Multivalency” as well as from the
Fonds der Chemischen Industrie (FCI). We thank Ilona Papp for
preliminary experimental contributions.
Supporting information for this article is available on the WWW
http://dx.doi.org/10.1002/anie.200801514.
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Scheme 2. Synthetic strategy for the Staudinger cyclization of peptides a) with and b) without the presence of side chain protecting groups.
tions,[17] and applications, including site-specific immobiliza-
tion of peptides on surfaces[18] and the ligation of protected
(glyco-)peptide fragments.[19] However, the chemoselectivity
of the traceless Staudinger ligation has only rarely been
addressed,[18a,19c,20] in contrast to the “bioorthogonal” Stau-
dinger ligation, which was developed by Bertozzi et al. for the
chemoselective functionalization of metabolically engineered
azidoglycans in cellular environment.[21] Along those lines, the
“traceless” Staudinger ligation has yet not been applied in the
ligation of unprotected peptide fragments.
In our strategy for the synthesis of cyclic peptides we
planned to use a borane-protected phosphinothiol to install
the reactive moieties for the Staudinger ligation—azide and
phosphine—in the same molecule (Scheme 1B).[20,22] After
borane removal under basic conditions[23] an intramolecular
version of the traceless Staudinger ligation can occur, which
was previously demonstrated for the synthesis of lactams and
biaryl-containing natural product analogues. By employing
the concept of an induced traceless Staudinger ligation, we
envisioned two possible synthetic strategies towards cyclic
peptides 1, both starting from the protected bifunctional
azidopeptide phosphinothioester 2. In the first route the
cyclization of 2 is initiated by borane cleavage in the presence
of side-chain-protecting groups to deliver the protected cyclic
peptide 3, and thus a final depro-
published protocols, was complete at 408C with DABCO.[20,24]
For an intramolecular application in peptide cyclization, we
focused on the solid-phase peptide synthesis (SPPS) of a
bifunctional azidopeptide phosphinothioester 2. Residues 1–
11 of Microcin J25 (GAGHVPEYFVG) were chosen as the
model sequence, which resembles the terminal circular loop
of
this
microbial
peptide.[26]
Standard
9-
fluorenylmethoxycarbonyl(Fmoc)-based SPPS using azido-
glycine 5 in the final coupling step on an acid-sensitive trityl
resin (TGT resin) and cleavage with 0.5% TFA in CH2Cl2
containing 2.5% TIS delivered the C-terminal-deprotected
peptide 6 (Scheme 3). This peptide was transformed into 2 by
activation with DIC/DMAP in CH2Cl2. HPLC and HRMS
(ESI-TOF) analysis (see Figure 1A and the Supporting
Information) demonstrated full conversion to 2, even when
only 1.1 equivalents of phosphinothiol 7, which was obtained
by deprotection of 4,[24] was used. The base-induced Stau-
dinger cyclization of 2 under optimized conditions was
performed at 7 mm concentration; the cyclized peptide 3
was obtained in very good overall conversion with only
minimal amounts of oxidized side product (see the Support-
ing Information).
In addition to this head-to-tail cyclization of a protected
peptide sequence by a base-induced traceless Staudinger
tection is required (Scheme 2a).
The second route utilizes a che-
moselective amide bond ring
closure in a globally deprotected
peptide for direct access to the
cyclized peptide 1 without addi-
tional chemical transformations
or deprotections (Scheme 2b).
The previously employed
conditions used for the borane
deprotection of a phosphino-
Scheme 3. Staudinger cyclization of peptides by base-induced borane cleavage : SPPS synthesis of the
bifunctional azidopeptide phosphinothioester 2. Reagents and conditions: a) 1. Fmoc-Xaa-OH or 5,
HBTU/HOBt, DIPEA, NMP; 2. 20% piperidine, NMP; b) 0.5% TFA, 2.5% TIS, CH2Cl2, 2 h; c) 7
(1.5 equiv), DIC (3 equiv), DMAP (cat.), CH2Cl2, 8 h; d) DABCO (3 equiv), DMF, 408C 4 h then 258C,
36 h. HBTU=2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HOBt=N-
hydroxybenzotriazole, DIPEA=diisopropylethylamine, NMP=N-methyl-2-pyrrolidone, TFA=trifluoro-
acetic acid, TIS=triisopropylsilane, DIC=diisopropylcarbodiimide.
thioester (708C) rendered the
generally applicability in poly-
peptide ligations difficult. How-
ever, deprotection of the borane-
protected acetylphosphinothiol
4, which can be synthesized by
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Scheme 4. Model studies for the intermolecular traceless Staudinger
ligation following borane removal under acidic conditions. Reagents
and conditions: a) TFA, 1 h, then TFA removal under high vacuum,
1 h; b) benzylazide (1 equiv), DABCO (2 equiv), [D7]DMF, 258C.
rates for the Staudinger ligation as residual TFAwould lead to
protonation of the iminophosphorane, which would poten-
tially enhance the electrophilicity of phosphorus and favor the
Staudinger reduction to the amine.
The use of acidic conditions for the deprotection has
several benefits over the base-induced version: The depro-
tection can be performed at room temperature, the reactive
borane species is converted into a chemically less reactive
borate compound, and bases milder than DABCO can be
used during the reaction sequence. However, and most
importantly for peptide Staudinger ligations, TFA-mediated
deprotection of an SPPS-derived azidopeptide phosphino-
thioester 2 leads to the simultaneous removal of all the
peptide side chain protecting groups, which would allow an
amide bond formation in the presence of unprotected side
chains.
The latter advantage was demonstrated on the azidopep-
tide phosphinothioester 2 (Scheme 5B), which was treated
with a solution containing 97.5% TFA and 2.5% TIS to yield
the deprotected azidopeptide phosphoniumthioester 10. Pep-
tide 10, which was characterized by HPLC and HRMS (ESI-
ToF) analysis (Figure 1B and the Supporting Information),
Figure 1. HRMS (ESI-TOF) analysis of the bifunctional azidopeptide
thioester 2 (A) and of the deprotected azido-peptide-phosphonium-
thioester 10. (B). C) HRMS (ESI-TOF) analysis and HPLC profile of
purified cyclic peptide 1a.
cyclization, we decided to further develop a system that made
use of the chemoselective features of the Staudinger reaction
(Scheme 2b). For such a strategy, deprotection of the peptide
side chains as well as the borane removal is needed such that
additional deprotection of the cyclized product is not
required. We therefore decided to switch to less frequently
used acidic conditions for the removal of borane protecting
group.[27]
In model studies treatment of acetyl-protected phosphi-
nothiol 4 with neat TFA and subsequent removal of the
volatiles under high vacuum yielded the free phosphine 8
(Scheme 4). Detailed 1H, 31P, and 11B NMR investigations
confirmed complete borane cleavage and demonstrated the
full conversion of the tetracoordinate borane into a less
reactive three-valent borate species (see the Supporting
Information).[28] A test reaction for the Staudinger ligation,
in this case with benzyl azide[25] to yield N-benzylacetamide
(9), was performed. Optimized conditions delivered an
excellent conversion of up to 95% when DABCO or
DIPEA was added to the reaction mixture to scavenge
residual amounts of TFA (see the Supporting Information)
and when short deprotection times (1 h) were maintained to
prevent thioester cleavage resulting from extended exposure
to TFA. The addition of a base was pivotal for high conversion
Scheme 5. Staudinger cyclization of peptides following borane removal
under acidic conditions. A) Deprotection of the bifunctional azidopep-
tide phosphinothioester 2 by treatment with TFA and subsequent
Staudinger cyclization. B) Sequences of cyclic peptides 1a–c and yields
of isolated product after HPLC purification. Reagents and conditions:
a) 97.5% TFA, 2.5% TIS, 1 h, then precipitation from dry ethyl ether;
b) DIPEA (20 equiv), DMF, 12 h. For details concerning the HPLC and
HRMS analysis see the Supporting Information. [a] Yield of isolated
product after HPLC purification (over three steps, see text and the
Supporting Information).
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did not show any tendency to undergo the Staudinger
cyclization without the addition of a base. However, it was
difficult to purify peptide 10 by HPLC as undesired partial
oxidation of the phosphine was observed. We therefore
decided to directly initiate the Staudinger cyclization after
deprotection with TFA. After precipitation from dry, cold
ethyl ether, peptide 10 was diluted to a concentration of 7 mm
in dry DMF, and DIPEA was added to initiate the Staudinger
cyclization. Analysis of the crude HPLC profile revealed that
the main product of the ligation corresponds to the fully
deprotected cyclic peptide 1a as verified by HRMS analysis
(see the Supporting Information). Further LC-MS analysis
revealed the oxidized phosphinothioester peptide with an
intact azide at the N terminus as a minor peptidic side
product. Importantly, no by-products formed by intermolec-
ular ligation were observed. Finally, the cyclic peptide 1a
(cyclo-GAGHVEPYFVG) was purified by preparative
HPLC and characterized by HRMS (Figure 1C). As no
intermediate HPLC purification between individual steps in
the synthesis was performed, an overall yield of 36% was
determined for the whole three-step synthetic scheme,
including the thioester synthesis, global deprotection, and
final cyclization.
Experimental Section
Synthesis of the protected bifunctional azidopeptide phosphino-
thioester 2: The azidopeptide was synthesized on an ABI 433a
peptide synthesizer using standard Fmoc-based SPPS conditions
(Fast-moc protocol with HBTU/HOBt activation). As the solid
support, TGTresin (Novabiochem) was used 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 removed 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, 1.5 equiv of borane-protected
diphenylphosphinomethanethiol (7) was added and the reaction
mixture was stirred for 12 h. The conversion to the corresponding
phosphinothioester 2 was determined by HPLC and HRMS analysis
(see the Supporting Information).
Deprotection of 2 with TFA and subsequent Staudinger cycliza-
tion: The crude reaction mixture containing the protected phopshi-
nothioester 2 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 10 was precipitated from 10 mL dry ethyl ether, charac-
terized by HPLC and HRMS methods (see the Supporting Informa-
tion), and redissolved in dry DMF (5 mL/0.033 mmol).
For the cyclization 20 equiv DIPEA was added to the reaction
mixture and the reaction mixture was stirred for 12 h. Cyclic peptide
1a was purified by preparative HPLC. HPLC and HRMS analysis
were carried out to confirm the identity of the final products and the
peptide intermediates.
This protocol was furthermore applied to the cyclization
of two other 11-residue peptides: Peptide 1b contained an
additional hydroxy functionality and one side chain amide
(sequence of 1b: GGIVPQFYSAG). The overall conversion
and side products were similar to those in the synthesis of 1a,
and the yield of isolated 1b was 31% (Scheme 5B). Finally,
peptide 1c containing the remaining residues of the Micro-
cin J25 sequence (GIGTPISFYGG, peptide 1c) was obtained
in 20% yield.
For HPLC and HRMS analysis of peptides 1a–c, 2, 3, and 10 see
Figure 1 and the Supporting Information. For the NMR anaylsis
concerning the TFA deprotection of 4 to give 8 as well as references
regarding synthetic procedures, see the Supporting Information.
Received: March 31, 2008
Published online: July 4, 2008
In summary, we have developed a method for the
synthesis of cyclic peptides employing two strategies based
on induced Staudinger ligation. Our approach offers an
efficient route to protected and unprotected peptide phos-
phinothioesters starting with an acid-labile trityl resin.
Particularly noteworthy is the fact that borane removal
under acidic conditions allows a chemoselective peptide
ligation in the presence of functional peptide side chains,
therefore broadening the general scope of the Staudinger
ligation in (poly-) peptide ligations as no Cys residues are
needed for the amide bond formation. Along those lines, this
strategy has the advantage that a concluding deprotection
step with the final peptide product is not necessary. Although
not all amino acid functionalities have been probed, this study
demonstrates the potential of the traceless Staudinger ligation
as a chemoselective amide bond forming reaction in the
synthesis of naturally occurring peptide substrates.
The cyclization in the presence of unprotected side chains
could take advantage of a structural preorganization of the
termini, thus favoring an intra- over an intermolecular amide
bond formation.[9] Studies concerning this structural influence
in combination with peptide concentration on the cyclization
yield, the development of chemoselective intermolecular
peptide Staudinger ligations, and the cyclization of larger
polypeptide sequences in aqueous solvents are currently
underway in our laboratory.
Keywords: chemoselectivity · cyclization · peptides ·
protecting groups · protein design
.
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