A photocleavable auxiliary for extended native chemical ligation

Article abstract of DOI:10.1002/ejoc.201500033

To extend the scope of native chemical ligations beyond X-Cys connections, auxiliaries that contain thiol moieties were developed to mimic the function of the Cys residue in the ligation reaction/capture step. Auxiliaries known so far feature complicated

Full text of DOI:10.1002/ejoc.201500033

FULL PAPER
DOI: 10.1002/ejoc.201500033
A Photocleavable Auxiliary for Extended Native Chemical Ligation
Christina Nadler,[a] André Nadler,[a] Christine Hansen,[a] and Ulf Diederichsen*[a]
Keywords: Peptides / Photochemistry / Cleavage reactions / Chemical ligations / Sulfur
To extend the scope of native chemical ligations beyond X-
Cys connections, auxiliaries that contain thiol moieties were
developed to mimic the function of the Cys residue in the
ligation reaction/capture step. Auxiliaries known so far
feature complicated attachment protocols and generally
need harsh conditions for their cleavage. Herein, we present
the development of a new photoremovable ligation auxiliary,
which is easily synthesized and attached to peptides that
contain a variety of N-terminal amino acids by a reductive
amination reaction. Ligations at Gly-Gly and Ala-Gly junc-
tions were carried out with high conversion, and the auxiliary
can be cleaved from the final product by using a mild photol-
ysis protocol. This methodological advancement is an impor-
tant step forward in peptide ligation chemistry.
opment. Arguably, the most promising approach in this re-
gard is the application of ligation auxiliaries positioned at
the N-terminal end of the C-terminal fragment, which then
undergo an NCL-like ligation reaction (Scheme 1). These
auxiliaries contain a thiol moiety, which mimics the func-
tion of the cysteine residue in NCL, and some can be
cleaved after ligation to thus generate the native peptide
bond.[14,15] So far, a number of different auxiliaries have
been introduced to allow ligation reactions at sites other
Introduction
Since its introduction in 1994, native chemical ligation
(NCL) has been the standard technique to synthesize or
synthetically modify proteins and large peptides by con-
necting two fragments with a native peptide bond.[1,2] The
technique crucially relies on rapid reversible thiol exchange
reactions that thioesters undergo whenever free thiols are
present. This reaction constitutes the first step of a ligation
sequence, connecting a peptide fragment that contains a C-
terminal thioester with the thiol group of an N-terminal
cysteine from a second fragment. In the second step, an
irreversible intramolecular rearrangement through an S–N
acyl shift takes place resulting in the formation of the native
peptide bond. The reaction is chemoselective and can also
be used in the semisynthesis of proteins by using intein-
mediated protein splicing to form recombinant thioester
peptides (expressed protein ligation, EPL).[3] Despite its ex-
tensive use in the synthesis of proteins, the applicability of
NCL is restricted by the need for a cysteine residue at the
N-terminus of the C-terminal peptide or protein fragment.
As Cys is one of the least prevalent amino acids in protein
sequences, this restriction gravely limits the use of NCL.[4]
Different methods have been developed to allow ligation
at sites other than X-Cys, such as post-ligational desulfur-
ization,[5–8] alkylation,[9] and the use of different chemo-
selective reactions in the capture step (i.e., the Staudinger
ligation).[10–13] Although these techniques have proven to
be of great synthetic value, they also have their inherent
limitations that still need to be addressed by further devel-
[a] Institut für Organische und Biomolekulare Chemie, Georg-
August-Universität Göttingen,
Scheme 1. Concept of auxiliary-mediated ligation. In the first step
(capture), the thioester function on one peptide undergoes a thiol
exchange with the thiol group of the auxiliary. After rearrangement
through an S–N acyl shift, the auxiliary can be cleaved to yield the
product, which contains the natural peptide bond.
Tammannstraße 2, 37077 Göttingen, Germany
E-mail: udieder@gwdg.de
http://www.diederichsen.chemie.uni-goettingen.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500033.
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than X-Cys. However, this impressive synthetic feat comes
with a caveat. Many of these auxiliaries cannot be cleaved
from the ligation product or require lengthy synthetic prep-
arations and attachment protocols.[15,16] Furthermore,
cleavage conditions are often harsh, which thus complicates
the synthesis of relatively labile compounds such as glyco-
peptides. To address the latter problem, a photocleavable
auxiliary was developed that enables ligations at Gly-Gly
and Ala-Gly ligation sites, but this method still requires a
lengthy synthesis and features a complicated attachment
protocol.[17,18] Ideally, an NCL auxiliary should be photo-
cleavable and accessible by a short synthesis. Its attachment
to the N-terminus of resin-bound peptides should be
straightforward, and it should be stable under acidic pept-
ide cleavage and deprotection conditions. Herein, we report
the synthesis and application of a photocleavable NCL
auxiliary, which meets the above-mentioned criteria.
Figure 1. Peptide that contains new auxiliary 1 (bottom); con-
ceptuallytheauxiliarycombinesthephotocleavable2-mercapto-1-(2-
nitrophenyl)ethyl moiety (top left) and the acid-labile 2-mercapto-
benzyl auxiliary (top right).
The use of a benzaldehyde scaffold for this new auxiliary
allows for it to be easily attached to the N-terminus of pept-
ides that contain different terminal amino acids by re-
ductive amination.[22,23] The natural product o-vanillin (2)
was used as an inexpensive starting material for the brief,
three-step synthesis of auxiliary 1. The first two steps were
carried out by following the reported synthesis of the corre-
sponding phenylsulfonyl compound.[24] The free hydroxy
group was transformed into the corresponding tosylate 5 in
high yield (Scheme 2). The nitro group was subsequently
introduced by using concentrated HNO3 to give a mixture
Results and Discussion
Synthesis of Auxiliary 1
The design of 3-methoxy-6-nitro-2-mercaptobenzyl of regioisomers, which were easily separated by recrystalli-
auxiliary 1 is based on previously reported acid-labile 2- zation from acetone. The undesired 4-nitro-substituted side
mercaptobenzyl auxiliaries (Figure 1), which enable ligation product 3 was insoluble in the acetone, whereas target com-
reactions at numerous ligation junctions (e.g., Gly-Gly, Ala- pound 4 dissolved. The thiol group was introduced by a
Gly, Lys-Gly).[19,20] The introduction of a nitro group at the nucleophilic aromatic substitution using trityl thiol, which
ortho position of the benzene ring should render the allowed for the isolation of the final product 1 in moderate
auxiliary photocleavable, as o-nitrobenzyl groups have been yields, presumably because of the steric demands of the
widely used as photoremovable protecting groups.[21] The bulky trityl group. The trityl group was chosen as a protect-
only photocleavable ligation auxiliary known so far also ing group for the thiol function because it is acid-labile and
contains an o-nitrobenzyl moiety, but its structure is other- easily cleaved under standard acidic peptide cleavage and
wise based on the 2-mercapto-1-phenylethyl auxiliaries, deprotection conditions, thus eliminating the need for a
which feature a complicated attachment protocol.[17,18]
dedicated deprotection step for the auxiliary.
Scheme 2. Synthesis of auxiliary 1 (Tos = 4-tolylsulfonyl, Trt = triphenylmethyl, NMP = N-methyl-2-pyrrolidone).
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A Photocleavable Auxiliary for Extended Native Chemical Ligation
Attachment of Auxiliary 1 to Peptides
By using a reductive amination reaction, auxiliary 1 was
attached to a number of peptides that contain different N-
terminal amino acids (Scheme 3). To prevent side reactions,
all reactions were conducted with the exclusion of light un-
der an inert atmosphere and using activated molecular
sieves to ensure complete removal of the residual water.
Peptides were synthesized by a standard 9-fluorenylmethyl-
oxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS)
on preloaded Wang resins. After removal of the final Fmoc
protecting group from the peptide, the resin was swollen in
anhydrous dichloromethane (DCM) for 1 h, and
auxiliary 1 and NaBH(OAc)3 were added in excess. The sus-
pension was agitated for 24 h and diluted with an excess
amount of DCM. Because of its low density, the resin could
be removed by pipette from the liquid surface. Afterwards,
the peptide was cleaved from the resin by using a mixture of
trifluoroacetic acid (TFA), triethylsilane (TES), and water
(95:2.5:2.5) simultaneously cleaving all the other protecting
groups including the trityl group on the thiol function of
the auxiliary. Cleavage times were limited to no more than
30 min, after which time the TFA was quickly removed by
evaporation to avoid decomposition of the auxiliary.
Figure 2. LC–MS traces of isolated auxiliary peptide 12 before and
after dissolution in a buffer solution that contained the reducing
agent tris(2-carboxyethyl)phosphine (TCEP), which indicates that
the oxidized form of the auxiliary peptide is readily reduced under
NCL conditions. Absorption was measured at 400 nm.
analysis of the reduced and oxidized forms revealed that a
cyclization reaction that involved the NH and SH moieties
was the most compelling explanation for the observed
formal H2 elimination (see Supporting Information).
To investigate the scope of the attachment protocol, we
examined the functionalization of a variety of peptides with
different N-terminal amino acids. Gly, Ala, Leu, Lys, and
Val were chosen as the N-terminal amino acids of peptides
to be functionalized with 1, as they exhibited different
grades of polarity and steric hindrance at the ligation junc-
tion in the following experiments. Auxiliary 1 was attached
to peptides that contained an N-terminal Gly, Ala, Leu, or
Lys in good overall yields with regard to the original resin
loading. The attachment of 1 to a peptide with an N-
terminal Val residue was possible, but only in low yields,
which indicates that auxiliary 1 is better suited for the func-
tionalization of non-β-branched N-terminal amino acids.
Scheme 3. Attachment of auxiliary 1 to peptides.
Apart from the expected product, the analysis of the ob-
tained peptides also showed the formation of a molecule
with a mass of two hydrogen atoms less than expected,
which corresponded to a formal elimination of H2. This
oxidized form accumulated upon prolonged storage. How-
ever, this oxidation turned out to be irrelevant to the actual
ligation, as the reducing conditions for the ligation reaction
led to nearly complete regeneration of the reactive reduced
form directly upon dissolution (Figure 2). Thus, the
auxiliary peptides were isolated and characterized in their
Ligation Reactions
Peptide thioesters for ligation reactions were synthesized
oxidized form and reduced in situ for the ligation reactions. on safety-catch sulfamylbutyryl resin by following known
With regard to the nature of the oxidized form of the literature protocols.[25,26] Thioester peptides with a Gly or
auxiliary peptides, the most obvious explanation would be Ala as the C-terminal amino acid were prepared as alkyl
the formation of a dimeric compound with a disulfide thioesters by using ethyl 3-mercaptopropionate and as aryl
bridge. However, this was ruled out by mass spectrometric thioesters by using (4-mercaptophenyl)acetic acid (MPAA).
analysis (see Supporting Information). To investigate the Alkyl thioesters are known to be less reactive in thiol ex-
structure of the oxidized species further, a small molecule changes, and therefore in ligation reactions, than aryl thio-
analogue (S2) of the auxiliary peptides was synthesized esters.[1,27,28] In agreement with this, the ligation reactions
by coupling auxiliary 1 to glycine methyl ester. NMR between alkyl thioesters and peptides that contained auxil-
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C. Nadler, A. Nadler, C. Hansen, U. Diederichsen
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iary 1 did not yield any product, whereas the formation of
product was possible by using aryl thioesters. As aryl thio-
esters are more prone to hydrolysis, even at low tempera-
tures, and thus can only be stored for a limited amount of
time, they were typically generated in situ by using the more
stable alkyl thioesters and an excess amount of the aryl
thiol (MPAA) as an additive during the ligation reactions.
A variety of ligation junctions were selected to show the
applicability and scope of auxiliary 1 (Table 1). Ligation
was possible at all chosen junctions, albeit with different
turnover. The ligation reactions were performed under ar-
gon and with the exclusion of light to prevent the oxidation
of the auxiliary peptides as well as any light-induced side
reactions and premature cleavage of the auxiliary. Ligation
buffer solutions contained 20 mm TCEP as a reducing agent
and were degassed prior to use. By following the reactions
with LC–MS at specific times, the conversion rate was de-
termined by comparing the LC peak areas of the product
to the auxiliary peptide at 400 nm (Figure 3). The auxiliary
chromophore absorbs at this wavelength to allow for the
visualization of both the auxiliary peptide and auxiliary-
containing product. All other components of the reaction
mixture were not visible at a wavelength of 400 nm.
Table 1. Overview of ligation reactions with auxiliary 1.
Ligation Conditions
junction
Thioester
peptide[a]
Auxiliary
peptide[b]
Conv.
[%]
Gly-Gly pH = 7.6, r.t.,
24 h
Gly-Ala pH = 7.0, r.t.,
24 h
Gly-Leu pH = 7.7,
50 °C,
ALYRG-SRЈ
(7)
ARVTLYGG-SRЈ
(8)
ARVTLYGG-SR
(10)
Aux-GFKV
(13)
Aux-AAFIKA
91
13
6
(9)
Aux-LAFIKA
(11)
Figure 3. (a) Ligation reaction at a Gly-Gly site between auxiliary
peptide 13 and thioester peptide 7. (b) LC–MS traces of the reac-
tion showing 91% conversion into product 15 after 24 h. Absorp-
tion was measured at 400 nm (GuHCl = guanidinium hydro-
chloride).
MPAA, 8 h
Ala-Gly pH = 7.1,
35 °C,
MPAA, 49 h
Ala-Ala pH = 7.0,
35 °C,
ARVTLYGA-SR
(14)
Aux-GYRA
(12)
82
5
ARVTLYGA-SR
(14)
Aux-AAFIKA
(9)
MPAA, 32 h
the reaction had a lower conversion of 13% after 24 h. The
ligation reaction of glycine thioester peptide 10 with an aux-
iliary peptide that contained an even more sterically de-
manding N-terminal amino acid such as Leu (11) provided
[a] R = ethyl 3-mercaptopropionate ester, RЈ = (4-mercaptophenyl)-
acetic acid. [b] Aux = auxiliary 1.
Several reaction parameters (temperature, reaction times, minimal product formation when conducted at room tem-
pH, concentration, use of preformed or in situ generated perature. When the reaction temperature was increased to
aryl thioester) were adjusted to each specific ligation reac- 50 °C, a conversion of 5% was detected after 8 h. However,
tion, as the reactivity varied at different ligation junctions. the thioester was completely hydrolyzed at this higher tem-
The employed conditions are specified in Table 1.
perature and length of time, and a higher conversion could
Reactions at the Gly-Gly ligation junctions proceeded not be attained. The reaction that featured sterically de-
smoothly with a high conversion of 91% within 24 h (Fig- manding Ala residues on both sides of the ligation site
ure 3). These findings were consistent with preformed aryl yielded a conversion of 5% after 32 h at an elevated tem-
thioester 7 and in situ generated thioester by using MPAA perature of 35 °C, which demonstrates that auxiliary 1 can
as an additive to the reaction mixture. To investigate the enable ligations at difficult junctions that were so far not
scope of the auxiliary 1 mediated ligation reactions, the accessible by using auxiliary-based ligation reactions.[15]
amino acid at the N-terminal end of the auxiliary peptide
In addition, we examined the exchange of the C-terminal
was varied with more sterically demanding amino acids Gly of the thioester peptide for the sterically more de-
than glycine. Ligations at a Gly-Ala site by employing thio- manding Ala. In the ligation reaction with Gly auxiliary
ester peptide 8 and auxiliary peptide 9 were possible, but peptide 12, a conversion of 82% was obtained after 49 h at
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A Photocleavable Auxiliary for Extended Native Chemical Ligation
35 °C. This longer reaction time is in line with the observa-
tion that Ala thioester peptides generally undergo slower
reactions than Gly thioester peptides in Native Chemical
Ligation reactions.[29]
Conclusions
A new photocleavable ligation auxiliary 1 was synthe-
sized and employed in NCL-like reactions at a variety
of ligation junctions. Auxiliary 1 was readily synthesized
in a three-step procedure and successfully introduced into
peptides that contain a wide array of different N-terminal
amino acids by reductive amination directly on the resin.
This straightforward protocol has several advantages com-
pared to the attachment protocols of previously known
ligation auxiliaries. It is a short, one-step procedure that is
fully compatible with acidic cleavage conditions and can be
easily applied to different N-terminal amino acids without
the need for different amino acid building blocks. Further-
more, the auxiliary peptides can be generated in good
yields.
Cleavage of the auxiliary was carried out by irradiation
with UV light (365 nm). Complete cleavage of auxiliary 1
was achieved after 40 min. The reaction did not produce
any side products, and the respective products could be
obtained with remarkably clean conversion (Figure 4).
Photolysis was possible by using samples taken directly
from the reaction mixture as well as purified samples. As
the auxiliary could be cleaved in all cases without any side
products, the limiting step seems to be the trans-
thioesterification and not the S–N acyl shift, as the cleavage
of the auxiliary from the nonrearranged intermediate would
yield the respective starting materials. A reason for this
might be the steric hindrance of the auxiliary as well as
the competition between the auxiliary thiol and the excess
amount of MPAA during transthioesterification. Increasing
the nucleophilicity of the thiol group of the auxiliary by
modifying its substitution pattern may be a solution to
improve ligation rates.
Auxiliary 1 enables ligation at Gly-Gly and Ala-Gly lig-
ation sites with high conversion. After the reaction, auxil-
iary
1 was readily cleaved from the final ligation
product by mild photolysis using UV light (365 nm). In
contrast to the previously reported photoremovable 2-
mercapto-1-(2-nitrophenyl)ethyl auxiliary,[17] ligations with
sterically more demanding amino acids at the N-terminus
of the auxiliary peptide and even with sterically demanding
amino acids on both sides of the ligation junction were
possible. However, the conversion was generally low (5–
13%). This hints at significant potential for the further de-
velopment of this methodology by generating derivatives
with different substitution patterns at the aromatic ring.
Modifying the nucleophilicity of the auxiliary thiol would
be a solution to increase ligation rates. This could be
achieved, for example, by introducing additional methoxy
groups on the phenyl ring, as those also increase the
nucleophilicity of the thiol group of 2-mercaptobenzyl
auxiliaries.[20,30] Continuous development of more sophisti-
cated peptide and protein ligation strategies is crucial to be
able to address challenging total syntheses or semisyntheses
of protein molecules. This is especially important when the
synthetic target features labile posttranslational modifica-
tions, and homogeneous material is required to establish
the precise role of an investigated protein in biochemical
experiments. The newly developed auxiliary 1 is an impor-
tant step in this direction, as it enables traceless NCL-like
reactions at ligation sites other than X-Cys and can be re-
moved by brief illumination with UV light, thus eliminating
the need for harsh deprotection conditions.
Experimental Section
General Methods: All of the employed reagents were of analytical
grade. Solvents were of the highest grade available. Dry solvents
were stored over molecular sieves (4 Å). All air- and water-sensitive
reactions were conducted under an inert atmosphere. For large-
scale reactions, the glassware was flame-dried, and for small-scale
reactions, which were conducted in tubes that were not heat
resistant, a purge and refill technique without heat was applied.
Purification by flash chromatography was conducted by using
Merck silica gel 60. The NMR spectroscopic data were recorded
with Varian instruments (Mercury 300, Unity 300, INOVA-500,
Figure 4. (a) Cleavage of auxiliary 1 from ligation product 16 by
irradiation with UV light (365 nm) for 40 min to form peptide 17.
(b) LC–MS traces of the cleavage reaction. UV absorption was
measured at 215 nm.
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C. Nadler, A. Nadler, C. Hansen, U. Diederichsen
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and INOVA-600). Chemical shifts are reported in ppm (for TMS,
δ = 0 ppm), and coupling constants JX,X are reported in Hz. ESI
3-Methoxy-6-nitro-2-(tritylthio)benzaldehyde (1): NaH (60% sus-
pension in mineral oil, 144 mg, 3.67 mmol, 1.00 equiv.) was added
n
mass spectra were recorded with Finnigan LCQ 7000 and Bruker
micrOTOF spectrometers. High resolution ESI spectra were ob-
tained with a Bruker APEX-Q IV 7T or Bruker micrOTOF spec-
trometer. LC–MS measurements were conducted on a Thermo Fin-
nigan LCQ ion trap mass spectrometer connected to a JASCO 851
autosampler with a Rheos 4000 pump and a Thermo Finnigan
Surveyor PDA (photodiode array) detector. Reactions were ana-
to a solution of triphenylmethanethiol (1.00 g, 3.67 mmol,
1.00 equiv.) in dry NMP (20 mL) at 0 °C under argon, and the mix-
ture was stirred at this temperature for 10 min. Compound 4
(1.29 g, 3.67 mmol, 1.00 equiv.) was added in portions, and the re-
action mixture was stirred for an additional 15 min. The reaction
was quenched by the addition of a saturated NH4Cl solution, and
the solution was diluted with Et2O (200 mL) and 1% HCl solution
(200 mL). The phases were separated, and the organic layer was
washed with H2O (3ϫ 200 mL) and brine (3ϫ 200 mL) and dried
with Na2SO4. The solvent was removed under reduced pressure,
lyzed on
a
Phenomenex Synergi LC Column, RP-C12
(150ϫ2.0 mm, 4 μm, 80 Å) or a MN Nucleodur EC column, RP-
C18, (100ϫ2.0 mm, 3 μm, 110 Å) with a linear gradient of eluent
A (H2O, 0.05% formic acid) to B (MeOH, 0.05% formic acid) in and after purification of the resulting residue by flash chromatog-
15 min. The conversions of the ligation reactions were calculated
by comparing the LC peak areas of the auxiliary peptide (starting
material) and the product that contained the auxiliary at 400 nm.
Origin[32] was used for all calculations employing a polynomial
baseline.
raphy (pentane/EtOAc, 2:1), 1 (519 mg, 1.47 mmol, 40%) was ob-
1
tained as a yellow solid. H NMR (301 MHz, CDCl3, 35 °C): δ =
3
9.53 (s, 1 H, CHO), 7.99 (d, JH,H = 9.2 Hz, 1 H, 5-H), 7.41–7.30
(m, 6 H, Trt-H), 7.30–7.12 (m, 9 H, Trt-H), 6.63 (d, 3JH,H = 9.2 Hz,
1 H, 4-H), 3.44 (s, 3 H, OCH3) ppm. 13CNMR (126 MHz, CDCl3,
27 °C): δ = 190.14 (CHO), 165.76 (C-3), 144.04 (C-6), 143.84 (Trt-
Cq), 139.14 (C-1), 130.16 (Trt-CH), 127.92 (C-5), 127.41 (Trt-CH),
127.10 (Trt-CH), 122.02 (C-2), 110.50 (C-4), 73.57 (SCPh3), 55.78
(OCH3) ppm. MS (ESI): m/z (rel. %) = 478.1 (100) [M + Na]+, 933
(93) [2M + Na]+. HRMS (ESI): calcd. for: [C27H21NO4SNa]+ [M
+ Na]+ 478.1083; found 478.1073; calcd. for [C27H20NO4S]– [M –
H]– 454.1119; found 454.1114. UV (MeCN): λmax = 229, 303 nm.
3-Methoxy-2-p-tolylsulfonylbenzaldehyde (5): o-Vanillin (2, 10.0 g,
65.8 mmol, 1.00 equiv.) and KOH (4.00 g, 70.1 mmol, 1.07 equiv.)
were dissolved in H2O (70 mL). Tosyl chloride (12.5 g, 65.8 mmol,
1.00 equiv.) was added, and the mixture was stirred at room tem-
perature for 1 h. DCM (5 mL) was added, and the precipitate was
removed by filtration, washed with H2O (3ϫ 100 mL), and dried
under reduced pressure to yield the crude product (19.0 g,
1
62.0 mmol, 94%), which was used without further purification. H
Peptide Synthesis: Except for the peptide thioesters, all peptides
were synthesized by manual Fmoc solid-phase peptide synthesis on
preloaded Wang resins by using O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-
tetramethyluronium hexafluorophosphate (HBTU)/ 1-hydroxy-
benzotriazole (HOBt)/N,N-diisopropylethylamine (DIPEA) as the
coupling agents and applying a fivefold excess amount of the acti-
vated Fmoc-protected amino acids in NMP. The last Fmoc group
was retained on the peptide for storage purposes and removed by
the standard protocol shortly before functionalization. The resins
were dried in vacuo overnight before functionalization.
NMR (300 MHz, CDCl3, 29 °C): δ = 10.09 (s, 1 H, CHO), 7.84–
7.71 (m, 2 H, Tos-HAr), 7.55–7.44 (m, 1 H, 5/6-H), 7.44–7.24 (m,
3 H, Tos-HAr and 5/6-H), 7.11 (dd, 3JH,H = 8.2 Hz, 4JH,H = 1.5 Hz,
1 H, 4-H), 3.59 (s, 3 H, OCH3), 2.47 (s, 3 H, Tos-CH3) ppm. 13C
NMR (126 MHz, CDCl3, 27 °C): δ = 188.10 (C=O), 152.72 (C-3),
145.85 (C-2), 132.79 (CTos-4), 131.34 (CTos-1), 129.79 (CTos-2/6),
128.74 (CTos-3/5), 128.00 (C-6), 119.54 (C-5), 118.08 (C-4), 56.06
(OCH3), 21.86 (Tos-CH3) ppm. MS (ESI): m/z (rel. %) = 307.1 (6)
[M + H]+, 329.1 (100) [M + Na]+, 635.1 (56) [2M + Na]+. HRMS
(ESI): calcd. for [C15H15O5S]+ [M + H]+ 307.0635; found 307.0638;
calcd. for [C15H14O5SNa]+ [M + Na]+ 329.0454; found 329.0455;
calcd. for [C15H13O5S]– [M – H]– 305.0489; found 305.0485.
Attachment of Auxiliary 1 to Peptides: The resin (20.0 mg, resin
load: 0.2–0.4 mmolg–1) was suspended in anhydrous DCM (2 mL)
3-Methoxy-6-nitro-2-(p-tolylsulfonyl)benzaldehyde (4): Benzalde- under argon over activated molecular sieves (3 Å). The suspension
hyde 4 was synthesized according to a reported protocol for the
corresponding phenylsulfonyl derivative.[24] 3-Methoxy-2-(p-tolyl-
sulfonyl)benzaldehyde (5, 18.1 g, 59.2 mmol, 1.00 equiv.) was added
to 90% HNO3 (42 mL) at 0 °C over the course of 30 min. After
additional stirring for 30 min, the mixture was poured onto ice
(240 g), and the mixture was warmed to room temperature. The
resulting precipitate was collected by filtration and washed with
H2O (50 mL). The solid was suspended in acetone, and was kept
at 4 °C for 20 h. The solvent was removed by filtration and the
crude product purified by flash chromatography (pentane/EtOAc,
was stirred for 1 h to allow the resin to swell. Auxiliary 1
(10.0 equiv.) was dissolved in anhydrous DCM (1 mL) under argon
and transferred to the suspension. After the addition of
NaBH(OAc)3 (25.0 equiv.), the suspension was stirred at room tem-
perature for 24 h with the exclusion of light. The suspension was
subsequently transferred into a test tube and diluted with DCM
(25 mL). Molecular sieves and the boron compounds were sepa-
rated from the resin by sedimentation (15 min). The resin, which
rose to the surface, was pipetted into a BD syringe, washed with
DCM (5ϫ 1 mL), and dried in vacuo overnight. A mixture of TFA/
4:1) to yield the product (12.6 g, 35.9 mmol, 60%) as a yellow solid. TES/H2O (95:2.5:2.5, 1 mL) was added to the dry resin, and the
1H NMR (300 MHz, CDCl3, 29 °C): δ = 10.08 (s, 1 H, CHO), 8.15 mixture was agitated at room temperature for 30 min with the ex-
3
(d, JH,H = 9.2 Hz, 1 H, 5-H), 7.86–7.75 (m, 2 H, Tos-HAr), 7.44– clusion of light. The solution was transferred into a lightproof
7.33 (m, 2 H, Tos-HAr), 7.07 (d, 3JH,H = 9.3 Hz, 1 H, 4-H), 3.78 (s, flask, and the resin was washed with TFA (2ϫ 1 mL). The solvent
3 H, OCH3), 2.49 (s, 3 H, Tos-CH3) ppm. 13C NMR (126 MHz, was removed under reduced pressure, and the resulting solid was
CDCl3, 27 °C): δ = 185.89 (CHO), 157.96 (C-3), 146.14 (CTos-4), suspended in cold Et2O (12 mL) and precipitated by centrifugation
139.45 (C-6), 135.15 (C-1), 132.49 (CTos-1), 131.93 (C-2), 129.81 at –20 °C and 9000 min–1 for 30 min. The solvent was decanted,
(CTos-2/6), 128.64 (CTos-3/5), 124.98 (C-4), 113.16 (C-5), 56.85 and the crude product was dissolved in a mixture of H2O and
(OCH3), 21.96 (Tos-CH3) ppm. MS (ESI): m/z (rel. %) = 352.1 (37)
MeCN (1:1). The resulting solution was lyophilized and purified
[M + H]+, 374.1 (95) [M + Na]+, 725.1 (100) [2M + Na]+. HRMS by reverse phase HPLC. The auxiliary peptides were obtained in
(ESI): calcd. for [C15H14NO5S]+ [M + H]+ 352.0485; found
352.0483; calcd. for [C15H13NO5SNa]+ [M + Na]+ 374.0305; found
isolated yields of up to 39% based on resin loading (included pept-
ide synthesis, reductive amination, cleavage from the resin, and
374.0305; calcd. for: [C15H12NO5S]– [M – H]– 350.0340; found HPLC purification). All products were isolated in their oxidized
350.0328.
form.
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 3095–3102

A Photocleavable Auxiliary for Extended Native Chemical Ligation
Aux-GFKV (13): MS (ESI): m/z (rel. %) = 645.27 (100) [Mox
+
overnight. (e) Cleavage of protecting groups: A solution of TFA,
TIS, and H2O (95:2.5:2.5, 8 mL) was added to the residue, and the
mixture was stirred at room temperature for 2 h. The solvent was
removed in vacuo. The crude peptide was suspended in cold Et2O
(10 mL), and the resulting solution was centrifuged (9000 rpm,
–10 °C, 20 min). The ether was decanted, and the precipitation pro-
cess was repeated. The crude peptide was lyophilized from H2O
and purified by reverse phase HPLC.
H]+. HRMS (ESI): calcd. for [C30H41N6O8S]+ [Mox
645.2701; found 645.2707.
+
H]+
Aux-AAFIKA (9): MS (ESI): m/z (rel. %) = 815.14 (100) [Mox
+
H]+. HRMS (ESI): calcd. for [C38H54N8O10S]+ [Mox + H]+
815.3756; found 815.3751.
Aux-LAFIKA (11): MS (ESI): m/z (rel. %) = 857.4 (100) [Mox
+
H]+. HRMS (ESI): calcd. for [C41H61N8O10S]+ [Mox + H]+
857.4226; found 857.4222.
ALYRG-SR (20): MS (ESI): m/z (rel. %) = 348.2 (100) [M +
2H]2+
, 695.3 (64) [M +
H]+. HRMS (ESI): calcd. for:
[C31H51N8O8S]+ [M + H]+ 695.3545; found 695.3541.
Aux-KAFIKA (18): MS (ESI): m/z (rel. %) = 436.7 (100) [Mox
+
2H]2+
, 872.4 (18) [Mox +
H]+. HRMS (ESI): calcd. for
ALYRG-SRЈ (7): MS (ESI): m/z (rel. %) = 365.2 (100) [M +
[C41H62N9O10S]+ [Mox + H]+ 872.4335; found 872.4337; calcd. for
[C41H64N9O10S]2+ [Mox + 2H]2+ 436.7204; found 436.7207.
2H]2+
, 729.4 (54) [M +
H]+. HRMS (ESI): calcd. for:
[C34H49N8O8S]+ [M + H]+ 729.3389; found 729.3387; calcd. for
[C34H47N8O8S]– [M – H]– 727.3243; found 727.3246.
Aux-GYRA (12): MS (ESI): m/z (rel. %) = 661.2 (100) [Mox
+
H]+, 683.2 (9) [Mox
+
Na]+. HRMS (ESI): calcd. for:
[C28H37N8O9S]+ [Mox + H]+ 661.2399; found 661.2398.
ARVTLYGG-SRЈ (8): MS (ESI): m/z (rel. %) = 985.5 (100) [M +
H]+, 1971.2 (4) [2M + H]+.
Aux-GAFIKA (19): MS (ESI): m/z (rel. %) = 801.4 (100) [Mox
+
H]+. HRMS (ESI): calcd. for: [C37H52N8O10S]+ [Mox + H]+
801.3600; found 801.3596.
ARVTLYGG-SR (10): MS (ESI): m/z (rel. %) = 476.75 (61) [M +
2H]2+
, 952.49 (100) [M +
H]+. HRMS (ESI): calcd. for:
[C42H71N11O12S]2+ [M + 2H]2+ 476.7497; found 476.7497; calcd.
for: [C42H70N11O12S]+ [M + H]+ 952.4921; found 952.4921.
Synthesis of Peptide Thioesters: Peptide thioesters were synthesized
on a 4-sulfamylbutyryl resin by following literature protocols.[25,26]
(a) Resin loading: The resin (2.00 g, approximately 1.34 mmol) was
suspended in CH3Cl (50 mL), which had been filtered over basic
allox prior to the reaction. For attachment of glycine, the resin was
suspended in N,N-dimethylformamide (DMF, 30 mL). The resin
was allowed to swell in the respective solvent for at least 30 min.
The respective Fmoc-protected amino acid (4 equiv.) was added,
and the mixture was stirred for 10 min and then cooled to –20 °C.
After 20 min of stirring at this temperature, benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP,
4 equiv.) and DIPEA (8 equiv.) were added. The mixture was
stirred at –20 °C for 8 h. For glycine, the reaction mixture was
warmed to room temperature overnight. For all of the other func-
tionalizations (because of the risk of racemization), the resin was
pipetted directly into a BD syringe, washed with DCM (3ϫ
10 mL), DMF (3ϫ 10 mL), DCM (3ϫ 10 mL), and MeOH (3ϫ
10 mL) and dried overnight under reduced pressure. The procedure
was repeated (2 or 3 times) until sufficient resin loading was
achieved. The loading density was determined by detecting the UV
absorption of dibenzofulvene, which formed upon Fmoc cleav-
age.[31] (b) Peptide synthesis on 4-sulfamylbutyryl resin: Attach-
ment of the Fmoc-protected amino acids was conducted by using
the standard Fmoc SPPS conditions mentioned above. The first
three amino acids were double coupled. For the introduction of
the last amino acid, a Boc-protected (Boc = tert-butoxycarbonyl)
derivative was used. The resin was washed with NMP and DCM
and dried in vacuo. Batchsize: 0.2 mmol. (c) Activation: Iodoace-
tonitrile (300 equiv.), which was filtered through basic
allox, was added to a solution of DIPEA (60.0 equiv.) in NMP
(8 mL). The mixture was agitated at room temperature for 22 h
with the exclusion of light. The solution was discarded, and the
resin was washed with NMP (5ϫ 10 mL), DMF (5ϫ 10 mL), and
DCM (5ϫ 10 mL) and dried in vacuo overnight. (d) Cleavage from
resin: The resin was swollen in DMF for 1 h. The respective thiol
[MPAA (RЈ) or 3-mercaptopropionic acid ethyl ester (R),
50.0 equiv.] and sodium thiophenolate (0.50 equiv.) were dissolved
in DMF/DCM (1:1, 10 mL), and the resulting suspension was agi-
tated at room temperature for 24 h with the exclusion of light. The
solution was collected, and the resin was washed with DMF (3ϫ
10 mL). The solvents were removed under reduced pressure, and
the residue was washed with cold ether and kept under vacuum
ARVTLYGA-SR (14): MS (ESI): m/z (rel. %) = 483.76 (12) [M +
2H]2+
, 966.51 (100) [M +
H]+. HRMS (ESI): calcd. for:
[C43H72N11O12S]+ [M + H]+ 966.5077; found 966.5071.
Ligation Experiments: All ligations were conducted under argon
with the exclusion of light by utilizing sealed black 1.5 mL centri-
fuge tubes. The protocol is based on a general ligation strategy that
is used in previously published extended Native Chemical Ligation
protocols.[14,20] Ligation buffers were prepared by using a filtered
stock solution of guanidium hydrochloride (6 m) and NaH2PO4
(100 mm), which could be stored at 4 °C for 1 month. The addition
of the additives [MPAA (50 mm), TCEP (20 mm)] and the adjust-
ment of the pH were performed directly before each ligation. All
buffers were degassed prior to application. The thioester and auxil-
iary peptide were weighed into a black centrifuge tube and set un-
der vacuum for 30 min. Afterwards, they were dissolved in the de-
gassed buffer solution under argon. To ensure complete dissol-
ution, the closed tubes were shaken, sonicated, and centrifuged.
The reaction mixtures were stirred at room temperature or in a
temperature-controlled oil bath. At consistent time intervals, sam-
ples for LC–MS measurements were removed under argon. For
this, a sample of the reaction solution (10 μL) was pipetted into an
opaque vial and diluted with a mixture of H2O and MeCN (1:1)
that contained 0.1% TFA (20 μL). After the indicated reaction
time, the product was purified using reverse phase HPLC.
ALYRGG(Aux)FKV (15): Auxiliary peptide 13 and thioester pept-
ide 7 at pH = 7.64 and room temp. for 24 h without MPAA yielded
15 (91%). MS (ESI): m/z (rel. %) = 604.8 (100) [M + 2H]2+, 1207.6
(19) [M + H]+.
ARVTLYGGA(Aux)AFIKA (21): Auxiliary peptide 9 and thioester
peptide 8 at pH = 6.99 and room temp. for 24 h without MPAA
yielded 21 (13%). MS (ESI): m/z (rel. %) = 818.3 (100) [M +
2H]2+
, 1634.6 (5) [M +
H]+. HRMS (ESI): calcd. for:
[C75H117N19O20S]2+ [M + 2H]2+ 817.9216; found 817.9235; calcd.
for [C75H116N19O20S]+ [M + H]+ 1634.8359; found 1634.8365.
ARVTLYGGL(Aux)AFIKA (22): Auxiliary peptide 11 and thio-
ester peptide 10 at pH = 7.66 and 50 °C for 8 h with MPAA as an
additive yielded 22 (6%). MS (ESI): m/z (rel. %) = 839.3 (100) [M
+ 2H]2+, 1676.7 (13) [M + H]+, 1698.6 (6) [M + Na]+.
Eur. J. Org. Chem. 2015, 3095–3102
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3101

C. Nadler, A. Nadler, C. Hansen, U. Diederichsen
FULL PAPER
[2] S. B. H. Kent, Chem. Soc. Rev. 2009, 38, 338–351.
[3] T. Muir, D. Sondhi, P. Cole, Proc. Natl. Acad. Sci. USA 1998,
95, 6705–6710.
ARVTLYGAG(Aux)YRA (23): Auxiliary peptide 12 and thioester
peptide 14 at pH = 7.10 and 35 °C for 49 h with MPAA as an
additive yielded 23 (82%). MS (ESI): m/z (rel. %) = 748.2 (100) [M
+ 2H]2+, 1494.5 (14) [M + H]+. HRMS (ESI): calcd. for:
[C66H101N19O19S]2+ [M + 2H]2+ 747.8615; found 747.8617; calcd.
for: [C66H100N19O19S]+ [M + H]+ 1494.7158; found 1494.7136.
[4] P. McCaldon, P. Argos, Proteins: Struct., Funct., Genet. 1988,
4, 99–122.
[5] L. Z. Yan, P. E. Dawson, J. Am. Chem. Soc. 2001, 123, 526–
533.
[6] D. Crich, A. Banerjee, J. Am. Chem. Soc. 2007, 129, 10064–
ARVTLYGAA(Aux)AFIKA (24): Auxiliary peptide 9 and thioester
peptide 14 at pH = 7.00 and 35 °C for 32 h with MPAA as an
additive yielded 24 (5%). MS (ESI): m/z (rel. %) = 824.8 (100) [M
+ 2H]2+, 1648.5 (26) [M + H]+.
10065.
[7] Q. Wan, S. J. Danishefsky, Angew. Chem. Int. Ed. 2007, 46,
9248–9252; Angew. Chem. 2007, 119, 9408.
[8] C. Haase, H. Rohde, O. Seitz, Angew. Chem. Int. Ed. 2008, 47,
6807–6810; Angew. Chem. 2008, 120, 6912.
[9] C. E. Hopkins, P. B. O. Connor, K. N. Allen, C. E. Costello,
D. R. Tolan, Protein Sci. 2002, 11, 1591–1599.
[10] E. Saxon, C. Bertozzi, Science 2000, 287, 2007–2010.
[11] E. Saxon, J. I. Armstrong, C. R. Bertozzi, Org. Lett. 2000, 2,
2141–2143.
ARVTLYGGG(Aux)AFIKA (16): Auxiliary peptide 19 and thio-
ester peptide 10 at pH = 7.60 and room temp. for 72 h with MPAA
as an additive yielded 16 (70%). MS (ESI): m/z (rel. %) = 810.9
(100) [M + 2H]2+, 1620.8 (13) [M + H]+. HRMS (ESI): calcd. for
[C74H115N19O20S]2+ [M + 2H]2+ 810.9138; found 810.9151; calcd.
for [C74H114N19O20S]+ [M + H]+ 1620.8203; found 1620.8208.
[12] M. Köhn, R. Breinbauer, Angew. Chem. Int. Ed. 2004, 43,
3106–3116; Angew. Chem. 2004, 116, 3168.
[13] J. Tailhades, N. Patil, M. A. Hossain, J. D. Wade, J. Pept. Sci.
2015, 21, 139–147.
Photocleavage: Samples of the ligation product or ligation reaction
mixture were pipetted onto a transparent, polystyrene six-well
tissue culture test plate and illuminated by using a Biostep UV
transilluminator (UST-20L-8E, 365 nm, filter size 20ϫ20 cm,
100% intensity). Full cleavage was generally reached after 40 min.
The reaction was analyzed by LC–MS using the same protocol as
that for ligation reactions. The product was isolated by reverse
phase HPLC.
[14] L. Canne, J. Steven, S. Kent, J. Am. Chem. Soc. 1996, 118,
5891–5896.
[15] C. P. R. Hackenberger, D. Schwarzer, Angew. Chem. Int. Ed.
2008, 47, 10030–10074; Angew. Chem. 2008, 120, 10182.
[16] D. Macmillan, Angew. Chem. Int. Ed. 2006, 45, 7668–7672; An-
gew. Chem. 2006, 118, 7830.
[17] C. Marinzi, J. Offer, R. Longhi, P. E. Dawson, Bioorg. Med.
Chem. 2004, 12, 2749–2757.
ARVTLYGGAAFIKA (25): Ligation product 21 was employed to
give 25. MS (ESI): m/z (rel. %) = 719.7 (100) [Mw/o Aux + 2H]2+
,
[18] T. Kawakami, S. Aimoto, Tetrahedron Lett. 2003, 44, 6059–
6061.
1437.7 (17) [Mw/o Aux + H]+.
[19] T. Kawakami, K. Akaji, S. Aimoto, Org. Lett. 2001, 3, 1403–
ARVTLYGGLAFIKA (26): Ligation product 22 was employed to
1405.
give 26. MS (ESI): m/z (rel. %) = 740.7 (100) [Mw/o Aux + 2H]2+
,
[20] J. Offer, C. Boddy, P. Dawson, J. Am. Chem. Soc. 2002, 124,
4642–4646.
[21] P. Klán, T. Solomek, C. G. Bochet, A. Blanc, R. Givens, M.
Rubina, V. Popik, A. Kostikov, J. Wirz, Chem. Rev. 2013, 113,
119–191.
[22] J. Offer, P. E. Dawson, Org. Lett. 2000, 2, 23–26.
[23] B. Wu, J. Chen, J. D. Warren, G. Chen, Z. Hua, S. J. Danishef-
sky, Angew. Chem. Int. Ed. 2006, 45, 4116–4125; Angew. Chem.
2006, 118, 4222.
[24] J. F. Biernat, J. S. Bradshaw, B. E. Wilson, N. K. Dalley, R. M.
Izatt, J. Heterocycl. Chem. 1986, 23, 1667–1671.
[25] B. J. Backes, J. A. Ellman, J. Org. Chem. 1999, 64, 2322–2330.
[26] R. Ingenito, E. Bianchi, D. Fattori, A. Pessi, J. Am. Chem. Soc.
1999, 121, 11369–11374.
1479.6 (3) [Mw/o Aux + H]+.
ARVTLYGAGYRA (27): Ligation product 23 was employed to give
27. HRMS (ESI): calcd. for: [C58H94N18O16]2+ [Mw/o Aux + 2H]2+
649.3542; found 649.3536.
ARVTLYGGGAFIKA (17): Ligation product 16 was employed to
give 17. MS (ESI): m/z (rel. %) = 712.4 (100) [Mw/o Aux + 2H]2+
,
1423.8 (15) [Mw/o
+
H]+. HRMS (ESI): calcd. for
Aux
[C66H108N18O17]2+ [Mw/o Aux + 2H]2+ 712.4064; found 712.4068;
calcd. for [C66H107N18O17]+ [Mw/o
1423.8059.
+ H]+ 1423.8056; found
Aux
[27] P. E. Dawson, M. J. Churchill, M. R. Ghadiri, S. B. H. Kent, J.
Am. Chem. Soc. 1997, 119, 4325–4329.
Acknowledgments
[28] E. C. B. Johnson, S. B. H. Kent, J. Am. Chem. Soc. 2006, 128,
6640–6646.
Financial support from the Deutsche Forschungsgemeinschaft
(DFG) (IRTG 1422, Metal Sites in Biomolecules and SPP 1623,
Chemoselective Reactions for the Synthesis and Application of Func-
tional Proteins) is gratefully acknowledged.
[29] T. M. Hackeng, J. H. Griffin, P. E. Dawson, Proc. Natl. Acad.
Sci. USA 1999, 96, 10068–10073.
[30] J. Offer, P. E. Dawson, Org. Lett. 2000, 2, 23–26.
[31] M. Gude, J. Ryf, P. White, Lett. Pept. Sci. 2002, 203–206.
[32] OriginPro 8 SR0, version v8.0724, OriginLab, Northampton,
MA.
[1] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science
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Received: January 10, 2015
Published Online: March 30, 2015
3102
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 3095–3102