Features of Auxiliaries That Enable Native Chemical Ligation beyond Glycine and Cleavage via Radical Fragmentation

Article abstract of DOI:10.1002/chem.201705927

Native chemical ligation (NCL) is an invaluable tool in the total chemical synthesis of proteins. Ligation auxiliaries overcome the requirement for cysteine. However, the reported auxiliaries remained limited to glycine-containing ligation sites and the acidic conditions applied for cleavage of the typically applied N-benzyl-type linkages promote side reactions. With the aim to improve upon both ligation and cleavage, we systematically investigated alternative ligation scaffolds that challenge the N-benzyl dogma. The study revealed that auxiliary-mediated peptide couplings are fastest when the ligation proceeds via 5-membered rather than 6-membered rings. Substituents in α-position of the amine shall be avoided. We observed, perhaps surprisingly, that additional β-substituents accelerated the ligation conferred by the β-mercaptoethyl scaffold. We also describe a potentially general means to remove ligation auxiliaries by treatment with an aqueous solution of triscarboxyethylphosphine (TCEP) and morpholine at pH 8.5. NMR analysis of a ¹³C-labeled auxiliary showed that cleavage most likely proceeds through a radical-triggered oxidative fragmentation. High ligation rates provided by β-substituted 2-mercaptoethyl scaffolds, their facile introduction as well as the mildness of the cleavage reaction are attractive features for protein synthesis beyond cysteine and glycine ligation sites.

Full text of DOI:10.1002/chem.201705927

A Journal of
Accepted Article
Title: Features of Auxiliaries that Enable Native Chemical Ligation
beyond Glycine and Cleavage via Radical Fragmentation
Authors: Simon Ferdinand Loibl, Andre Dallmann, Kathleen Hennig,
Carmen Juds, and Oliver Seitz
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To be cited as: Chem. Eur. J. 10.1002/chem.201705927
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Features of Auxiliaries that Enable Native Chemical Ligation
beyond Glycine and Cleavage via Radical Fragmentation
Simon F. Loibl, Andre Dallmann, Kathleen Hennig, Carmen Juds and Oliver Seitz*[a]
Abstract: The native chemical ligation (NCL) is an invaluable tool in
the total chemical synthesis of proteins. Ligation auxiliaries overcome
the requirement for cysteine. However, the reported auxiliaries
remained limited to glycine-containing ligation sites and the acidic
conditions applied for cleavage of the typically applied N-benzyl-type
linkages promote side reactions. With the aim to improve upon both
ligation and cleavage, we systematically investigated alternative
ligation scaffolds that challenge the N-benzyl dogma. The study
revealed that auxiliary-mediated peptide couplings are fastest when
the ligation proceeds via 5-membered rather than 6-membered rings.
Substituents in α-position of the amine shall be avoided. We observed,
perhaps surprisingly, that additional β-substituents accelerated the
ligation conferred by the β-mercaptoethyl scaffold. We also describe
a potentially general means to remove ligation auxiliaries by treatment
with an aqueous solution of triscarboxyethylphosphine (TCEP) and
morpholine at pH 8.5. NMR analysis of a 13C-labeled auxiliary showed
that cleavage most likely proceeds through a radical-triggered
oxidative fragmentation. High ligation rates provided by β-substituted
2-mercaptoethyl scaffolds, their facile introduction as well as the
mildness of the cleavage reaction are attractive features for protein
synthesis beyond cysteine and glycine ligation sites.
Introduction
Peptide ligation reactions are the key to the total chemical
synthesis of proteins. At current, the native chemical ligation
(NCL) [1] is providing the most reliable access to proteins up to a
size of 304 amino acids.[2] The reaction involves two unprotected
peptide segments i.e. a peptide thioester 1 and a cysteine peptide
2 (Scheme 1). At pH ≥ 6 thioesters undergo thiol exchange
reactions, which eventually leads to a thioester intermediate 3.
This sets the stage for an intramolecular S→N acyl shift yielding
the native peptide bond in the final ligation product 4. NCL
reactions proceed efficiently in aqueous solution at lower
millimolar concentration of peptides. However, the requirement
for N-terminal cysteine in the C-terminal peptide fragment limits
Scheme 1: Concepts of A) NCL and B) auxiliary-mediated peptide ligation. C)
the breadth of NCL chemistry. Target proteins may lack cysteine
Chemical structures of established (left) Nα-auxiliaries and novel base-labile Nα-
auxiliaries (MAP, 3-mercapto-2-aryl-propyl; MAIP, 2-mercapto-2’-aryl-isopropyl;
ME; 2-mercaptoethyl; MP, 2-mercaptopropyl: MPE, 2-mercapto-2-phenyl-ethyl).
or contain cysteine at positions that are difficult to access by NCL
chemistry such as Pro-Cys, Thr-Cys, Ile-Cys and Val-Cys
junctions.[3]
Ligation auxiliaries have been developed with the aim to
expand the applicability of NCL (Scheme 1B). This approach
relies on the N-terminal attachment of a cleavable scaffold that
offers a mercapto group in a position that facilitates the
intramolecular S→N acyl shift in thioester intermediate 3a.[4] The
pioneering work of Dawson[5] and Kent[6] focused on N-benzyl
type auxiliaries (6 and 7 in Scheme 1C) which were designed to
[a]
S. F. Loibl, Dr. A. Dallmann, K. Hennig, C. Juds, Prof. Dr. O. Seitz
Institut für Chemie
Humboldt-Universität zu Berlin
Brook-Taylor-Str. 2, 12489 Berlin (Germany)
E-mail: oliver.seitz@chemie.hu-berlin.de
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201705927
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enable cleavage by acidolysis[6-7] or photolysis[8]. However, the
rather low ligation rates at ligation junctions beyond glycine as
well as the vulnerability of the formed peptide bond to acid-
induced amide cleavage prevented a broad acceptance of this
ligation technology.
other studied auxiliaries are susceptible to cleavage under mildly
basic (pH 8.5), radical conditions. We also introduce a revised
mechanism for cleavage of the 2-mercapto-2-phenethyl auxiliary.
In the ligation-desulfurization method,[9] the mercapto group is
connected to the framework of the N-terminal amino acid. This
approach provides for fast ligation reactions and the advent of
metal-free radical desulfurization[10] sparked the development of a
variety of thiolated amino acid building blocks.[11] However, only 3
out of the 15 thiolated amino acids used in NCL reactions are
commercially available. As a result, the application of the ligation-
desulfurization approach is limited to labs skilled in organic
synthesis.
An ideal method would provide access to arbitrary ligation
sites without the efforts required for the preparation of a panel of
building blocks. Given the ease by which ligation auxiliaries are
introduced upon reductive amination in the last step of automated
solid-phase peptide synthesis, we reconsidered the design of
auxiliary scaffolds. We sought to i) improve ligation rates by
avoiding steric bulk at the ligation site and ii) prevent cleavage of
the formed N-alkyl amides by getting around the acidic cleavage
conditions that typically induce S→N acyl shift reactions. This
analysis guided us to the development of new auxiliary scaffolds
such as the 2-mercapto-2-phenethyl (MPE) auxiliary[12], which is
the first auxiliary that escapes the need for glycine at the ligation
junction and permits auxiliary removal under mild basic conditions
when S→N acyl shifts are favored[13] over N→S acyl shifts.
Previous reports stressed the importance of native chemical
Results and Discussion
Scaffold synthesis and introduction of auxiliaries in peptides
Native chemical ligation reactions involving the 2-mercaptoethyl
auxiliaries MAIP, MPE and ME will proceed through 5-membered
cyclic ligation intermediates. By contrast, the 3-mercaptopropyl
auxiliary MAP will lead to a 6-membered ring. For the coupling of
2-mercaptoethyl-type auxiliaries with
a C-terminal peptide
fragment we chose a reductive amination route (Scheme 2A).
Ketone 10 was envisioned as a suitable building block for the
introduction of the MAIP auxiliary. We commenced the synthesis
by allylation of 4-nitroaniline[15] and subsequent oxidation of the
terminal alkene[16] to the epoxide 8. Treatment with tert.-
butylmercaptane in presence of tetrabutylammonium fluoride
(TBAF) induced the regioselective opening of the epoxide. The β-
hydroxy thioether 9 was subsequently converted to the desired
ketone 10 by Dess-Martin oxidation.
ligation-type reactions proceeding through 5-membered ring
14]
transition states.[4-5,
However, despite a 6-membered ring
intermediate (3aI) the reactivity of mercaptobenzyl-type auxiliaries
7 seems comparable to the 2-mercapto-1-phenethyl auxiliaries 6.
The seemingly high reactivity was attributed to a template effect
conferred by the mercaptobenzyl system.[5] On the other hand,
steric bulk was identified as the main reason for the generally low
ligation rate obtained with the benzylic auxiliaries 6 and 7. We felt
that the reasons as to why some aminothiol systems react faster
in NCL-type chemistry than others seemed rather unclear.
To explore the requirements for a high reactivity in auxiliary-
mediated ligations we embarked on a comparative study of
different auxiliary scaffolds. Herein, we evaluate 4 different
ligation auxiliaries. By comparing the 3-mercapto-2-arylpropyl
(MAP) and 2-mercapto-2’-aryl-isopropyl (MAIP) auxiliaries with
the recently reported 2-mercapto-2-phenethyl (MPE) auxiliary we
shine a light on the role of i) the size of the ring formed in the NCL
transition state and ii) the substituent in α position to the amino
group (Scheme 1C). To facilitate reactions at sterically
demanding ligation sites we sought options to increase scaffold
flexibility at the N-attachment site. We therefore included the 2-
mercaptoethyl (ME) auxiliary in this study. A comparison of the
ME auxiliary with the 2-mercaptopropyl (MPE) auxiliary will
expose the role of substituents in the β position to the amine. Of
note, the ME scaffold was the first ligation auxiliary reported in the
seminal paper by Canne et al.[4] However, a method for the
removal of this simplest ligation auxiliary imaginable was still
lacking. We show that the mercaptoethyl group as well as the
Scheme 2: Synthesis of A) precursors for auxiliary introduction via B) reductive
amination. 1) tBuSH, TBAF, 79%; 2) Dess-Martin periodinane, DCM/tBuOH,
58%; 3) N, O HNMeOMe, DIPEA, DCM; 4) TrtSH, DIPEA, DCM, 71% (over two
steps); 5) LiAlH4, THF, toluene, 87%; 6) 10, NaCNBH3, AcOH, CH(OMe)3,
MeOH, DMF or 11/15, NaCNBH3, AcOH, iPrOH, NMP; 7) 2G-MAIP: TFA/TIS
then TFA/TFMSA/anisole, 20%; 2G-MPE: TFA/TIS, 25%; 2G-MPE: TFA/TIS,
18%. The letter “G” in 2G represents N-terminal glycine.
For introduction of the 2-mercapto-2-phenethyl-auxiliary MPE
via reductive amination we had recently reported the 6-step
synthesis of aldehyde 11. A similar building block (15) should
allow the introduction of the unsubstituted 2-mercaptoethyl
auxiliary (ME). Bromoacetylbromide was converted to the
Weinreb amide 13 prior to treatment with tritylmercaptane. The
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10.1002/chem.201705927
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reaction of 14 with lithiumaluminumtetrahydride afforded
aldehyde 15.
ligation reactions (Fig. 1D). Based on the reaction half-times, the
MAP scaffold provided 6-fold higher reactivity than the MAIP
scaffold (5AG-MAIP: t = 417 min, 5AG-MAP: t = 66 min).
Likewise, the MAP auxiliary afforded the sterically more
demanding Leu-Gly junction in >90 % yield within 24 h. In this
reaction, the MAIP auxiliary provided < 70% yield. We tested the
two auxiliaries in ligations beyond glycine junctions. However, the
reaction including alanine peptide thioester 1A and N-terminal
alanine auxiliary peptides 2A-MAP and 2A-MAIP furnished no
ligation product (see Fig. S14).
At the first glance, it seems surprising that auxiliary-mediated
ligation via 6-membered ring intermediates proceeded faster than
reactions via 5-membered intermediates. However, we assumed
that the results hint at the importance of the substituent in α-
position to the secondary amine. Usually, ring closure via a 5-exo-
trig process is preferred to a 6-exo-trig process. However, the 5-
exo-trig reaction with the MAIP auxiliary is penalized by the α-
substituent, which presumably hinders access to the secondary
amine. We reasoned that the 2-mercapto-2-arylethyl auxiliary
(MPE) should enable faster ligations than the 3-mercapto-2-
arylpropyl auxiliary (MAP). While both auxiliaries lack the α-
substituent the reaction on the MPE scaffold can proceed via the
preferred 5-exo-trig rather than the 6-exo-trig process. Indeed, the
2-mercapto-2-phenyl ethylauxiliary (MPE) provided the highest
reactivity and afforded Ala-Gly and Leu-Gly (Fig. 1E) junctions in
3-fold lower reaction half time than the 3-mercapto-2-arylpropyl
(MAP) auxiliary (Fig. 1D, Table 1). Most importantly, the reactivity
of the MPE auxiliary was sufficiently high to allow the formation of
sterically demanding ligation sites that did not contain glycine (Fig.
1I). After 24 h reaction time the MPE auxiliary afforded the Ala-
Asn and the Leu-Asn junction in 5AN-MPE and 5LN-MPE in 79 %
and 63 % yield, respectively (Table 1).
In the next step, we equipped the resin-bound peptides 16
with the auxiliaries MAIP, MPE and ME by means of reductive
amination with tBu-S-protected ketone 10 or aldehydes 11 and 15
(Scheme 2B). The aldehydes 11 and 15 were dissolved in a
mixture of N-methylpyrolidone (NMP) and methanol and were
allowed to react with peptide resin 16 in presence of NaCNBH3
and acetic acid. The reductive amination of ketone 10 proceeded
best when the solution of NaCNBH3 in DMF/methanol also
contained the condensing agent trimethylorthoformiate. After
reductive amination, acidolytic cleavage released the desired
auxiliary-peptides 2G-MAIP, 2G-MPE and 2G-ME in 18-25%
overall yield.
For the introduction of the 6-ring-forming MAP auxiliary, we
prepared glycine building block 19 (Scheme 3A). First, 4-
nitrophenylacetic acid (17) was treated with paraformaldehyde in
a decarboxylative double Mannich-type reaction. The N-alkylated
morpholine derivative was converted to the chloride 18 by
reaction with isobutyl chloroformiate. Following N-alkylation of
½
½
glycine tBu-ester,
a
TBAF-promoted addition of tert.-
butylmercaptane and ester acidolysis furnished the desired
auxiliary-glycine conjugate 19. The tBu-S-protected monomer 19
was coupled to the resin bound peptide (Scheme 3B) and
afforded the auxiliary peptide 2G-MAP (22% yield) after
TFA/TFMSA-treatment and HPLC-purification.
As shown previously, the MPE scaffold is the first auxiliary to
enable native chemical ligation beyond glycine.[12] We were
interested to elucidate the role of the β-aryl substituent and
examined ligation with the unsubstituted 2-mercaptoethyl
auxiliary. Perhaps surprisingly, the unsubstituted ME auxiliary
Table 1. Yields[a] and half times[b] of auxiliary-induced NCL reactions (see
Fig. 1A).
Junction
Gly-Gly
MAP
MAIP
MPE
ME
/
Scheme 3: Synthesis of A) precursors for auxiliary introduction via B) coupling
of a preformed amino acid building block. 1) morpholine, (CH2O)n, toluene, 47%;
2) iBuOCOCl, toluene, 76%; 3) GlyOtBu, DIPEA, DCM, 86%; 4) tBuSH, TBAF,
79%; 5) HCl, dioxane, 99%; 6) PyBOP, DIPEA, DMF; 7) TFA/TIS then
TFA/TFMSA/anisole, 22%.
96 % (2 h)
95 % (4 h)
97 % (2 h)
t ≈ 5 min
½
t
½
≈ 4 min
t
½
≈ 14 min
Ala-Gly
Leu-Gly
Ala-Asn
Leu-Asn
98 % (24 h)
≈ 66 min
81 % (24 h)
≈ 417 min
96 % (24 h)
t ≈ 25 min
½
/
t
½
t
½
94 % (24 h)
≈ 190 min
66 % (24 h)
≈ 480 min
96 % (24 h)
≈ 55 min
70 % (24 h)
t ≈ 75 min
½
Peptide ligations
t
½
t
½
t
½
Next we compared the reactivity of the auxiliary scaffolds in native
chemical ligation reactions involving the peptide thioesters 1G, 1A
and 1L (Fig. 1A, the letters G, A and L indicate the C-terminal
amino acid). The flexible γ-mercaptopropyl (MAP) auxiliary in 2G-
MAP enabled nearly quantitative formation of the Gly-Gly junction
No product
No product
No product
No product
79 % (24 h)
≈ 290 min
75 % (24 h)
t ≈ 550 min
½
t
½
63 % (24 h)
≈ 960 min
34 % (24 h)
t ≈ 2200 min
½
t
½
in 5GG within less than 1 hour (t = 4 min, Fig. 1B, Table 1). The
½
α-substituted β-mercaptoalkyl auxiliary in 2G-MAIP required 4
[a] based on HPLC analysis after time indicated in parenthesis; [b] estimated
from reaction time course.
hours (t = 14 min) to deliver >90% ligation product (Fig. 1C). The
½
reactivity difference became more pronounced in Ala-Gly-forming
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Figure 1: a) Auxiliary-mediated ligation reaction between peptide thioesters 1Z and auxiliary modified peptides 2X-Aux (the letters Z and X represent the C-terminal
amino acid of the peptide thioester and the N-terminal amino acid of the auxiliary peptide, respectively). UPLC analysis of Gly-Gly ligation with the B) MAP and C)
MAIP auxiliary. D) Time course of Gly-Gly, Ala- Gly and Leu-Gly ligation reactions using MAP, MAIP and MPE auxiliaries. UPLC analysis of Leu-Gly ligation on E)
MPE and F) ME auxiliaries (5LGMe*, double acylated product obtained upon reaction of 5LGMe with thioester 1L, see Fig S10A for structure). Time course of G)
Ala-Asn and Leu-Asn ligations using MPE and ME auxiliaries. Comparison of the H) time course and UPLC analysis of Ala-Asn-ligation on the I) MPE and J) MP
auxiliary. Conditions: 2-5 mM peptides, 20 mM TCEP, 100 mM Na2HPO4, 3 vol% PhSH, rt, pH 7.5.
proved less reactive than the MPE auxiliary. While there was little
difference in native chemical ligation at the Leu-Gly junction (Fig.
1F), the unsubstituted ME scaffold required approx. double as
much time as the MPE scaffold to afford the Ala-Asn and Leu-Asn
bonds in 5AN-ME and 5LN-ME. With the MPE auxiliary the Leu-
Asn junction was established in > 60 % yield after 24 h whereas
the ME-mediated reaction proceeded in 34% yield only.
The inferior reactivity of the unsubstituted ME auxiliary
seemed counter-intuitive. A closer inspection exposes a rather
linear behavior of the reaction time course (Fig. 1G), which
suggests that ligations at sterically demanding junctions bear
resemblance to a zero order process. Such a case may result
from a rate limiting intramolecular S→N acyl shift which proceeds
from
a small steady state concentration of the thioester
intermediate 3a; a notion which has been put forward already in
previous reports on auxiliary-mediated ligations. [7b, 14, 17] Indeed,
UPLC analyses show formation of the thioester intermediate,
which precedes the S→N acyl shift (Fig. S15). Since the S→N
acyl shift progresses via a cyclic intermediate we assumed that
under these circumstances the native chemical ligation can be
analyzed in terms of a cyclization reaction. According to the
Thorpe-Ingold effect cyclizations are accelerated when a ring
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hydrogen atom is substituted by more space filling groups.[18] To
substantiate that the reactivity difference between the MPE and
the ME auxiliary is due to the Thorpe-Ingold effect we evaluated
the 2-mercaptopropyl (MP) auxiliary (see SI for details on
synthesis). The time courses of the Ala-Asn ligation mediated by
the MPE and the MP auxiliaries were nearly superimposable (Fig.
1H). We conclude that, perhaps surprisingly, β-substitution of β-
mercaptoalkyl auxiliaries increases rather than decreases the rate
of auxiliary-mediated native chemical ligation.
oxygen would trigger a β-fragmentation which would lead to
styrene and an amide radical. However, in presence of Na2S2O8
as radical starter (Fig. 2D) we noticed the formation of an N-
formylpeptide species 4GGf. This is indicative of a C-C bond
scission and questioned the postulated cleavage mechanism.
To elucidate the fate of the auxiliary we analyzed the reaction
by NMR spectroscopy. To help identify auxiliary debris we
incubated an α,β-13C-labelled analogue of ligation product 5GG-
MPE (see SI for synthesis) with an aqueous solution (5% D2O in
water) of TCEP and morpholine and followed the reaction by
heteronuclear single quantum coherence (HSQC)-spectroscopy.
The reaction mixture was extracted with CDCl3. The UPLC
analysis showed that CDCl3 extraction lead to the disappearance
of peak 21 (compare Fig.2B with Fig. 2C). The 2D-HSQC spectra
(see Fig. 2E and Fig. 2F) of the two phases exposed four distinct
species in the lower field. With the help of reference materials the
two major components were identified as benzaldehyde 21 and
N-formyl-morpholine 22 and the two remaining species as traces
of formic acid (23) and N-formylated peptide (<5% according to
UPLC analysis in Fig. 2B). The formation of benzaldehyde was
also observable in 1D-1H-NMR measurements of the mixture
obtained after cleavage of non-labeled MPE. The signals of the
Auxiliary removal
The results of the ligation experiments exposed the MPE scaffold
as the most reactive ligation auxiliary. In previous work, we had
demonstrated that treatment of ligation products such as 5GG-
MPE with triscarboxyethylphosphine (TCEP) and morpholine at
pH 8.5 in aqueous buffer resulted in the quantitative removal of
the auxiliary (Fig. 2A,B). Based on the observation that 2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPO) − a persistent radical used
as radical scavenger – inhibited the cleavage reaction (Fig.
S13),[12] we assumed a radical pathway for auxiliary cleavage.
Initially, we had hypothesized that the benzylic radical obtained
upon TCEP-mediated radical desulfurization in presence of
Figure 2: A) Removal of the 13C-labeled 2-mercaptophenethyl auxiliary (MPE) from ligation product 5GG-MPE13C. UPLC-analysis of the reaction mixture obtained
upon incubation of 5GG-MPE13C (B), C)) or 5GG-MPE (D)) after B) 16 h, C) after extraction with CDCl3 and D) after 1h, when cleavage was performed in presence
of 100mM Na2S2O8. HSQC-NMR spectra of the E) aqueous and F) organic phase after extraction of the reaction mixture with CDCl31.H-NMR spectra of 5GG-MPE
G) before and H) after cleavage. I) Comparison of NMR spectra obtained from the CDCl3 extract (red curve) and benzaldehyde in CDCl3 (blue curve) as authentic
reference.
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MPE auxiliary experienced a downfield shift upon cleavage
(compare Fig. 2G and Fig. 2H). Inspection of the organic phase
obtained after CDCl3 extraction revealed a pattern characteristic
of a phenyl ring system (Fig. 2I, red curve). Most notable was a
signal at δ = 9.97 ppm, which hinted at benzaldehyde. The
comparison of the 1H-NMR spectra with authentic reference
material (Fig. 2I, blue curve) confirmed the presence of
benzaldehyde in the reaction mixture.
Based on these results, we revise the postulated mechanism
for cleavage of the 2-mercapto-2-phenyl ethyl auxiliary (Scheme
4). Under basic conditions, the thiolate should be readily
converted to the thiyl-radical by air-oxidation.[19] The high
concentration of phosphine triggers the desulfurization[10] and
furnishes the benzyl radical 26. In the absence of a radical
scavenger (such as the high concentration of mercaptanes used
in radical desulfurization[10, 20]) benzyl radicals should rapidly react
with molecular oxygen dissolved in the aqueous buffer. Such
reactions are barrierless and proceed with very high speed.[21] As
described by Buckler[22] and Howard[23] the resulting peroxyradical
27 should readily react with TCEP under formation of the alkoxy-
radical intermediate 28, which based on literature evidence[24] is
expected to undergo β-fragmentation by release of
benzaldehyde.[24b, 25]
are accelerated upon use of radical initiator (Fig. 2D). In the last
step, morpholine (or hydroxide anions) attack the N-formylpeptide
at the most accessible electrophilic position i.e. the formyl carbon
leading to the native peptide 4 and N-formyl morpholine 22 (or
formiate 23).
The repeated involvement of oxygen raises the question as to
how the reaction would proceed when oxygen is limiting. Auxiliary
cleavage was performed at reduced concentration of oxygen (Fig.
S22) and we noticed the formation of an N-methylated peptide by-
product, which suggests that the amidoalkyl radical 29 can be
scavenged by hydrogen atom abstraction. The postulated
mechanism of auxiliary cleavage also provides an explanation for
the occurrence of N-terminally formylated fragment 24, which we
observed when the auxiliary was removed from 5GG-MPE (see
Fig. 2E). In principle, any auxiliary-modified fragment 2G-MPE
that remains after the ligation reaction will be converted to the N-
formyl species upon auxiliary cleavage. The N-terminal
formamide is not reactive enough to undergo aminolysis at pH 8.5.
However, despite our efforts to remove remaining auxiliary
peptide 2G-MPE, we still observed the formation of < 5% N-
terminal formamide 24. We therefore assume that the side-
reaction points to a peculiarity of formylated Xxx-Gly amide bonds.
Due to the absence of an α-substituent at glycine, the nuclophilic
attack of the bisacyl amide may occur also at the backbone. For
all other ligation junctions we expect that nucleophilic attack will
almost exclusively occur at the less hindered formyl group.
The mechanism in Scheme 4 describes a potentially generic
approach to remove ligation auxiliaries under mildly basic
conditions. Indeed, both the MAIP and the MAP auxiliaries were
removed by treatment of the ligation products 5GG-MAIP and
5GG-MAP, respectively, with the TCEP/morpholine mixture (Fig.
3A). The HPLC analysis suggested that the β-mercaptopropyl
scaffold of the MAIP auxiliary (Fig. 3B) reacted more smoothly
than the γ-mercaptopropyl scaffold of the MAP auxiliary (Fig. 3C).
We assume that the cleavage of the MAP auxiliary proceeds
through three alkoxy radical intermediates and scission of two
carbon-carbon bonds. This rather lengthy path probably is prone
to interference. Interestingly, the radical oxidative fragmentation
outlined in Scheme 4 also offered a means to remove the simplest
of all auxiliaries i.e. the mercaptoethyl (ME) scaffold. Cleavage of
the ME group from the Gly-Gly ligation product proceeded as
rapidly as cleavage of the MPE group. However, the noisy base-
line in the UPLC trace (Fig. 2D) suggests that cleavage of the ME
auxiliary results in more by-products than cleavage of the MPE
auxiliary. A slightly higher purity of the crude material was
obtained in cleavage reactions of the 2-mercapto-2-propyl (MP)
auxiliary (Fig. 3E). Though not optimized, the removal of the ME
but not the MPE auxiliary from Gln-Phe and Ala-Asn bonds
proved problematic (Fig. S21). Given that the MPE auxiliary
provides for the highest yields in both ligation and cleavage
reactions we consider the MPE auxiliary as the current gold
standard in auxiliary mediated native chemical ligation.
O
O
O
O
N
N
N
N
O2
HS
S
O
O
27
26
25
Ph
Ph
Ph
Ph
R3P R3PS
R3P
R3PO
O
O
O
O
O2
N
N
N
N
O
O
O
O
O
Ph
29
30
28
31
R3PO R3P
Ph
21
O2
O
O
+
R
2NCHO
N
N
H
22
R2NH
O
4
32
Scheme 4. Proposed cleavage mechanism of the 2-mercaptophenethyl (MPE)
auxiliary induced by TCEP and morpholine.
The remaining amidoalkyl radical 29 is a known species[26]
which likely is processed into the α-alkoxy radical 31 by means of
the reaction with molecular oxygen and subsequent
deoxygenation by TCEP. Without a connected downhill process
β-fragmentation of this radical (to an amide radical and
formaldehyde) should be disfavored. Rather, the formation of the
formyl species 22 and 23 indicates that the amidoalkoxy radical
31 undergoes oxidation to the N-formyl peptide 32 (such as 4GGf
in Fig. 2). This intermediate is observable when radical reactions
Conclusions
In this work we have extended the repertoire of auxiliary-mediated
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10.1002/chem.201705927
Chemistry - A European Journal
FULL PAPER
peptide ligation by introducing four new auxiliaries, which can be
in presence of a radical scavenger such as TEMPO suggests a
radical pathway of auxiliary removal. To elucidate the apparently
general mechanism we analyzed the cleavage of the MPE
auxiliary, the synthetic utility of which had been proven in the total
synthesis of the 48- and 126 amino acid long proteins opistoporin-
2[12] and mucin-1[27]
. NMR analysis of cleavage reactions
performed with isotopically labeled auxiliary revealed
benzaldehyde and formylmorpholine as the major components of
auxiliary debris. This and the observation of amide-linked formyl
peptide intermediates points to an oxidative fragmentation
reaction which most likely involves the attack of dissolved oxygen
on the alkyl radical formed upon radical desulfurization. Owing to
the high concentration of TCEP the resulting alkyl peroxyradicals
will rapidly be converted to alkoxyradicals. The cleavage
mechanism probably proceeds through two types of
alkoxyradicals with distinct reactivity. One type undergoes β-
fragmentation and therefore provides an aldehyde along with a
new alkylamido radical on the remaining auxiliary fragment
shortened by one carbon unit. Upon reaction with oxygen and
TCEP this radical gives rise to the second type of alkoxy radical,
which in the absence of an energetically accessible β-
fragmentation pathway will react with oxygen under formation of
an amide-linked formyl group. The latter is subject to aminolysis
(or hydrolysis) which eventually affords the auxiliary-free peptide.
The combination of radical oxygenation, phosphine induced
cleavage of O-O bonds and subsequent β-fragmentation or
oxidation of the resulting alkyloxy radicals suggests a potentially
generic means for the cleavage of amide-linked mercaptoalkyl
auxiliaries. According to that, the mercapto group serves as the
entry point for the induction of the radical oxidation-fragmentation
cascade which eventually results in the formation of a base-labile,
amide-linked formyl peptide. The mechanism plausibly explains
why such different auxiliaries as 3-mercapto-2-arylpropyl, 2-
Figure 3. A) Treatment with TCEP/morpholine as a generic method for inducing
cleavage of ligation auxiliaries. UPLC analysis of cleavage of the B) MAIP
auxiliary; C) MAP auxiliary; D) ME auxiliary and E) MP auxiliary. Conditions:
5GG-MAIP and 5GG-MAP: 0.5 mM peptides, 20 mM TCEP, 140 mM
morpholine pH 9-9.5, 40°C; 5GG-ME and 5GG-MP: 0.5 mM peptides, 100 mM
TCEP, 400 mM morpholine pH 8.5, 40°C.
mercapto-2’-aryl-isopropyl,
2-mercapto-2-phenethyl,
2-
mercaptopropyl and even 2-mercaptoethyl scaffolds are
cleavable by exposure to aqueous TCEP and morpholine.
The high ligation rate provided by the β-substituted 2-
mercaptoethyl scaffolds, their facile introduction by means of
reductive amination as well as the mildness of the cleavage
reaction, which seems particularly attractive for the synthesis of
posttranslationally modified peptides, are appealing features for
protein synthesis beyond cysteine and glycine ligation sites.
However, we are aware of potential shortcomings. Auxiliary
cleavage will affect ligation-unrelated cysteine side chains. We
have previously demonstrated that methionine residues are
tolerated and that side chain protecting groups such as the
acetamido (ACM) group protect cysteine side-chains from side
reactions.[12] A potential problem may arise from the requirement
for oxygen in the auxiliary cleavage reaction. In our experiments,
oxygen was made available by performing the experiments at
small micromol scale in plastic vials. At large scales oxygen may
be limiting and it may therefore prove necessary to explore
alternative sources of oxygen.
cleaved under mildly basic conditions in presence of TCEP. The
comparison of 5 different scaffolds suggests that extended native
chemical ligation is limited to glycine containing ligation sites as
long as the reaction proceeds through 6-membered rather than 5-
membered rings and auxiliaries contain substituents in α-position
of the amine. The study included the reactivity assessment of
three structurally similar ligation auxiliaries that were based on α-
unsubstituted β-mercaptoethyl scaffolds. We observed, perhaps
surprisingly, that additional β-substituents increased the ligation
rates. For example, the 2-mercapto-2-phenethyl (MPE) and 2-
mercaptopropyl (MP) auxiliaries formed an Ala-Asn junction at a
2-fold higher rate than the structurally simplest mercaptoethyl
(ME) auxiliary. We attribute the increased reactivity to the Thorpe
Ingold effect, which may gain importance when the S→N acyl shift
becomes rate limiting.
Even though the usefulness of the MPE auxiliary has been
demonstrated previously,[12] the two key observations i.e. i) β-
substituents increase ligation rate and ii) TCEP/morpholine
A notable observation was: all of the studied ligation
auxiliaries were susceptible to cleavage with an aqueous mixture
of TCEP and morpholine at pH 8.5. The supression of cleavage
cleaves ligation auxiliaries through
a
radical oxidation-
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10.1002/chem.201705927
Chemistry - A European Journal
FULL PAPER
Aldehyde 15: To a solution of weinreb amide 14 (250 mg, 662 µmol) in
dry THF (5 ml) was added dropwise LiAlH4 (207 µL, 725 µmol, 3.5 M in
THF/toluene) at -78°C. After 30 min NaHSO4 solution (25 ml, 5 wt%) was
added and the mixture was allowed to warm to room temperature. The
mixture was diluted by addition of DCM (50 ml), the organic phase was
separated, washed with sat. NaHSO4 solution (2 x 50 ml, 5 wt%), dried
over MgSO4 and concentrated in vacuo. The desired product (183 mg, 575
µmol, 87%) was isolated as brown oil after purification by flash-
fragmentation cascade suggest means for potentially improved
designs of cleavable ligation auxiliaries. In ongoing work we
deliberately avoid α-substituents and rather focus on variations of
β-substituents, which on the one hand may enable faster ligation
reactions and on the other hand allow tuning of the speed of
radical formation and radical fragmentation. We expect that β-
substituted β-mercaptoethyl scaffolds will become a highly useful
tool for the rapid total synthesis of (posttranslationally modified)
proteins by native chemical ligation beyond cysteine and glycine.
chromatography on silica-gel using cyclohexane
→ cyclohexane/
ethylacetate/EDMA (2/1/0.01) as a mobile phase. 1H-NMR (500 MHz,
CDCl3): δ = 8.77-8.76 (t, J = 2.5 Hz, 1H), 7.38-7.35 (m, 3H), 7.24-7.16 (m,
12H), 3.02-3.01 (d, J = 2.5 Hz, 2H). 13C-NMR (126 MHz, CDCl3): δ = 196.7,
144.2, 129.7, 128.3, 127.3, 67.6, 42.6.
Experimental Section
Allyl chloride 18: To a suspension of 4-nitrophenylacetic acid 17 (10.0 g,
55.2 mmol) and paraformaldehyde (4.01 g, 132 mmol) in toluene (25 ml)
was added morpholine (4.8 ml, 4.75 g, 55. 6 mmol). The reaction mixture
was heated (90°C, 1 h→ 100°C, 4 h) and subsequently concentrated in
vacuo. The residue was dissolved in DCM (50 ml), the organic phase
washed with water (2 x 25 ml) and concentrated in vacuo. The desired
morpholine derivative (6.34 g, 25.6 mmol, 47%) was isolated as a yellow
solid after purification by flash-chromatography on silica-gel using
cyclohexane/ethylacetate (3/1) as a mobile phase. 1H-NMR (500 MHz,
CDCl3): δ = 8.18-8.16 (dd, J1 = 9.0 Hz, J2 = 0.5 Hz, 2H), 7.71-7.68 (m, , J1
= 9.0 Hz, J2 = 2.5 Hz, 2H) 5.64 (d, J = 1.0 Hz, 1H) , 5.41 (d, J = 1.0 Hz,
1H), 3.66-3.64 (t, J = 4.5 Hz, 4H), 3.35 (d, J = 0.5 Hz, 2H), 2.46-2.45 (t, J
= 4.5 Hz, 4H). 13C-NMR (126 MHz, CDCl3): δ = 146.7, 142.4, 127.3, 123.6,
119.3, 67.1, 63.5, 53.5. HRMS (ESI): m/z = 249.1235 (C19H24NO5S2
(M+H)+, calc.: 249.1239); C13H16N2O3: 248.28 g·mol−1.To a solution of the
morpholine derivative (2.85 g, 11.5 mmol) in toluene (5 ml) was added
dropwise isobutyl chloroformate (1.8 ml, 1.89 g, 13.8 mmol). After 18 h the
reaction mixture was concentrated in vacuo. The desired product (1.73 g,
8.77 mmol, 76%) was isolated as a yellow oil after purification by flash-
chromatography on silica-gel using cyclohexane/ethylacetate (3/1) as a
mobile phase. 1H-NMR (500 MHz, CDCl3): δ = 8.17-8.15 (dd, J1 = 8.5 Hz,
J2 = 1.0 Hz, 2H), 7.62-7.60 (d, J = 8.5 Hz, 2H), 5.69 (s, 1H), 5.62 (s, 1H),
4.48-4.47(d, J = 0.5 Hz, 2H). 13C-NMR (126 MHz, CDCl3): δ = 147.4, 144.0,
Synthesis of auxiliary building blocks:
Alcohol 9: The epoxide 8 [16] (869 mg, 4.85 mmol) was dissolved in neat
tert.-butylmercaptane (1.10 ml, 880 mg, 9.75 mmol) and a catalytical
amount of TBAF*3H2O (27 mg, 0.25 mmol) was added. The reaction
mixture was stirred at 50°C for 18 h and subsequently concentrated in
vacuo. The desired product (1.03 g, 3.81 mmol, 79%) was isolated as a
yellow oil after purification by flash-chromatography on silica-gel using
cyclohexane/ethylacetate (4/1) as a mobile phase. 1H-NMR (500 MHz,
CDCl3): δ = 8.14-8.11 (m, J1 = 9.0 Hz, , J2 = 2.5 Hz, J3 =2.0 Hz, 2H), 7.41-
7.39 (m, J1 = 9.0 Hz, , J2 = 2.5 Hz, J3 =2.0 Hz, 2H), 3.94-3.89 (m, 1H),
2.96- 2.93 (m, J1 = 13.5 Hz, , J2 = 4.5 Hz, 1H) 2.89-2.85 (m, J1 = 14.0 Hz, ,
J2 = 8.0 Hz, 1H), 2.76-2.72 (m, J1 = 13.0 Hz, , J2 = 4.5 Hz, 1H), 2.62 (s,
1H), 2.58-2.54 (m, J1 = 13.0 Hz, , J2 = 7.5 Hz, 1H), 1.29 (s, 9H). 13C-NMR
(126 MHz, CDCl3): δ = 146.8, 146.3, 130.4, 123.6, 70.6, 42.8, 42.5, 35.9,
31.1. HRMS (ESI): m/z = 270.1160 (C13H20NO3S (M+H)+, calc.: 270.1164);
C13H19NO3S: 269.35 g·mol−1
.
S-tBu-protected ketone 10: To a solution of the alcohol 9 (969 mg, 3.60
mmol) in a mixture of DCM/tert.butanol (22 ml, 9/1; v/v) was added Dess-
Martin periodinane (12.6 g, 4.43 mmol, 15 wt% solution in DCM). After 3.5
h the reaction was stopped by addition of a mixture of aqueous NaHCO3
(10 wt%, 10 ml) and aqueous NaHSO3 (saturated, 10 ml). The aqueous
phase was separated and extracted with ethylacetate (2x 20 ml). The
combined organic phases were dried over MgSO4 and subsequently
concentrated in vacuo. The desired product (558 mg, 2.09 mmol, 58%)
was isolated as a yellow solid after purification by flash-chromatography
on silica-gel using cyclohexane/ethylacetate (4/1) as a mobile phase. 1H-
NMR (500 MHz, CDCl3): δ = 8.16-8.14 (m, 2H), 7.39-7.37 (m, 2H), 4.06 (s,
2H), 3.36 (s, 2H), 1.30-1.29 (m, 9H). 13C-NMR (126 MHz, CDCl3): δ =
203.6, 147.1, 141.7, 130.7, 123.8, 46.4, 43.9, 39.5, 30.8. HRMS (ESI): m/z
= 266.0856 (C13H16NO3S (M-H)-, calc.: 266.0845); C13H17NO3S: 267.34
142.3, 127.0, 123.7, 120.3, 45.9. HRMS (ESI): m/z
= 220.0138
(C9H8ClNO2Na (M+Na)+, calc.: 220.0141); C9H8ClNO2: 197.62 g·mol−1
.
MAP-auxiliary glycine conjugate 19: To a solution of the allyl chloride
18 (435 mg, 2.20 mmol) in DCM (10 ml) was added subsequently glycine
tert-butyl ester hydrochloride (1.11 g, 6.60 mmol) and DIPEA (1.13 ml, 1.50
g, 11.7 mmol). After 16 h the reaction mixture was washed with (2 x 10 ml),
dried over MgSO4 and concentrated in vacuo. The desired glycine
intermediate 1 (555 mg, 1.90 mmol, 86%) was isolated as a yellow oil after
purification
by
flash-chromatography
on
silica-gel
using
g·mol−1
.
cyclohexane/ethylacetate (4/1) as a mobile phase. 1H-NMR (500 MHz,
CDCl3): δ = 8.19-8.16 (dt, J1 = 9.0 Hz, J2 = 2.5 Hz, 2H), 7.65-7.62 (dt, J1 =
9.0 Hz, J2 = 2.5 Hz, 2H), 5.57 (s, 1H), 5.44 (d, J = 1.0 Hz, 1H), 3.68 (d, J
= 1.0 Hz, 2H), 3.30 (s, 2H), 1.77 (s, 1H), 1.46 (s, 9H). 13C-NMR (126 MHz,
CDCl3): δ = 171.7, 147.3, 146.4, 144.4, 127.1, 123.8, 117.6, 81.5, 52.9,
50.8, 28.2. HRMS (ESI): m/z = 293.1496 (C15H21N2O4 (M+H)+, calc.:
293.1501); C15H20N2O4: 292.33 g·mol−1. The glycine intermediate1 (494
mg, 1.69 mmol) was dissolved in neat tert.-butylmercaptane (238 µL, 190
mg, 2.11 mmol) and a small amount of TBAF·3H2O (27 mg, 0.09 mmol)
was added. The reaction mixture was stirred at rt for 16 h and concentrated
in vacuo. The desired intermediate 2 (508 mg, 1.34 mmol, 79%) was
isolated as a yellow oil after purification by flash-chromatography on silica-
gel using cyclohexane/ethylacetate (4/1) as a mobile phase. 1H-NMR (500
MHz, CDCl3): δ = 8.18-8.16 (d, J = 3.5 Hz, 2H), 7.41-7.39 (d, J = 3.5 Hz,
2H), 3.27-3.19 (dd, , J1 = 17.0 Hz, J2 = 6.5 Hz, 2H), 3.10-3.04 (m , 2H)
2.98-2.94 (m, 2H), 2.91-2.87 (m, 2H), 2.76-2.72 (m, 2H), 1.51 (s, 1H), 1.42
(s, 9H), 1.25 (s, 9H). 13C-NMR (126 MHz, CDCl3): δ = 171.5, 150.4, 147.0,
Weinreb amide 14: To an ice-cold solution of N, O-dimethylhydroxylamine
hydrochloride (0.50 g, 5.13 mmol) in DCM (50 ml) was added DIPEA (1.33
g, 1.79 ml, 10.3 mmol) and dropwise bromoacetylbromide 12 (1.04 g, 446
µL, 5.13 mmol). The mixture was allowed to warm to room temperature
and stirred for 2.5 h. The organic phase was washed with sat. NaHSO4
solution (2 x 15 ml), dried over MgSO4 and concentrated in vacuo. The
residue was dissolved in DCM (25 ml). To this solution was added
triphenylmethyl mercaptan (1.70 g, 6.15 mmol) and DIPEA (0.66 g, 0.89
ml, 5.10 mmol). After 16 h the mixture was concentrated in vacuo. The
desired product (1.37 g, 3.62 mmol, 71%; C23H23NO2S; MW = 377.50
g·mol−1
) was isolated as white solid after purification by flash-
chromatography on silica-gel using cyclohexane/ethyl-acetate/EDMA
1
(2/1/0.01) as a mobile phase. H-NMR (300 MHz, CDCl3): δ = 7.50-7.46
(m, 6H), 7.35-7.21 (m, 9H), 3.48 (s, 3H), 3.13 (s, 3H), 3.10 (s, 2H).
This article is protected by copyright. All rights reserved.

10.1002/chem.201705927
Chemistry - A European Journal
FULL PAPER
128.8, 123.9, 81.4, 54.0, 51.8, 46.9, 42.5, 32.3, 30.9, 28.2. HRMS (ESI):
m/z = 383.1998 (C19H31N2O4S (M+H)+, calc.: 383.2005); C19H30N2O4S:
382.52 g·mol−1. For removal of the tBu ester intermediate 2 (2.40 g, 6.27
mmol) was dissolved in 4 M HCl in dioxane (5 ml). The reaction mixture
was stirred at rt for 2 h and subsequently concentrated in vacuo. The
product was co-evaporated with toluene (3 x 5 ml). The desired MAP-
glycine conjugate (2.25 g, 6.20 mmol, 99%) was obtained as a white solid.
1H-NMR (500 MHz, CD3OD): δ = 8.28-8.26 (d, J = 8.5 Hz, 2H), 7.63-7.61
(d, J = 8.5 Hz, 2H), 3.88- 3.80 (m, J1 = 17.0 Hz, , J1 = 8.0 Hz, 2H), 3.63-
3.50 (m, J1 = 9.5 Hz, J2 =5.0 Hz, 2H), 3.41-3.35 (m, J = 7.5 Hz, 1H), 3.01-
2.86 (m, J1 = 7.5 Hz, J2 =5.0 Hz 2H), 1.29 (s, 9H). 13C-NMR (126 MHz,
CD3OD): δ = 168.9, 149.2, 148.1, 130.6, 125.1, 52.7, 45.1, 43.6, 33.2, 31.2.
HRMS (ESI): m/z = 327.1374 (C15H23N2O4S (M+H)+, calc.: 327.1379);
5GG-MAIP: UPLC: tR = 2.7 min (3-40% B in 4 min); ESI-MS: 1663.6
((M+H)+, calc.: 1663.8), 832.5 ((M+2H)2+, calc.: 832.4), 555.5 ((M+3H)3+
,
calc.: 555.4); C73H110N22O21S: 1663.9 g·mol−1. 5GG-MAP: UPLC: tR = 2.9
min (3-40% B in 4 min); ESI-MS: 1663.7 ((M+H)+, calc.: 1663.8), 832.5
((M+2H)2+, calc.: 832.4), 555.5 ((M+3H)3+, calc.: 555.4); C73H110N22O21S:
1663.9 g·mol−1. 5AG-MAIP: UPLC: tR = 2.7 min (3-40% B in 4 min); ESI-
MS: 1677.6 ((M+H)+, calc.: 1677.8), 839.5 ((M+2H)2+, calc.: 839.4); 560.2
((M+3H)3+, calc.: 559.9); C74H112N22O21S 1677.9 g·mol−1. 5AG-MAP:
UPLC: tR = 3.0 min (3-40% B in 4 min); ESI-MS: 1677.5 ((M+H)+, calc.:
1677.8), 839.5 ((M+2H)2+, calc.: 839.4); 560.2 ((M+3H)3+, calc.: 559.9);
C74H112N22O21S 1677.9 g·mol−1. 5LG-MAIP: UPLC: tR = 7.1-7.3 min (3-
40% B in 10 min); ESI-MS: 1720.8 ((M+H)+, calc.: 1719.9), 860.5
((M+2H)2+, calc.: 860.4); 574.2 ((M+3H)3+, calc.: 574.0); C77H118N22O21S
1712.0 g·mol−1. 5LG-MAP: UPLC: tR = 7.3-7.5 min (3-40% B in 10 min);
ESI-MS: 1719.8 ((M+H)+, calc.: 1719.9), 860.5 ((M+2H)2+, calc.: 860.4);
574.2 ((M+3H)3+, calc.: 574.0); C77H118N22O21S: 1712.0 g·mol−1. 5LG-ME:
UPLC-MS: tR = 2.9 min (3-40% B in 4 min); m/z = 793.4 ((M+2H)2+, calc.:
C15H23ClN2O4S: 362.87 g·mol−1
.
Synthesis of auxiliary peptides
792.9), 529.2 ((M+3H)3+, calc.: 528.9); C70H113N21O19S: 1584.9 g·mol−1
.
The peptide 16 was synthesized on a Rink-amide resin (Fmoc-strategy,
see SI.). The N-terminal Fmoc-group was removed prior to reductive
amination or coupling. After reaction times indicated the tBu-protected
auxiliary peptides were cleaved from the peptide-resin with a mixture of
TFA/TIS (95/5, v/v), precipitated in ether and treated with a mixture of
TFA/TFMSA/anisole (8/1/1, v/v/v) at 0°C to liberate the mercapto-groups
(deprotection of tBu-thioether, see Fig. S4 and S6)). The desired auxiliary
peptides were purified by semi-preparative HPLC (gradients of solvent B
(0.1% TFA, 1% H2O, 98.9% acetonitrile) in solvent A (0.1% TFA, 1%
acetonitrile, 98.9% H2O) and isolated as white solids after lyophilisation.
5AN-MPE: UPLC-MS: tR = 3.8 min (3-30% B in 8 min); m/z = 800.5
((M+2H)2+, calc.: 800.4), 534.1 ((M+3H)3+, calc.: 533.9); C69H110N22O20S:
1599.8 g·mol−1. 5AN-ME: UPLC-MS: tR = 3.8 min (3-30% B in 6 min); m/z
= 839.1 ((M+2H)2+, calc.: 838.4), 559.7 ((M+3H)3+, calc.: 559.2.6);
C75H114N22O20S: 1675.9 g·mol−1. 5LN-MPE: UPLC-MS: tR = 4.4 min (3-
30% B in 8 min); m/z = 821.8 ((M+2H)2+, calc.: 821.4), 548.2 ((M+3H)3+
calc.: 548.0); C72H116N22O20S: 1641.9 g·mol−1. 5LN-ME: UPLC-MS: tR
,
=
3.8 min (3-30% B in 6 min); m/z = 860.2 ((M+2H)2+, calc.: 859.4), 573.6
((M+3H)3+, calc.: 573.3); C78H120N22O20S: 1718.0 g·mol−1. 5AN-MP UPLC-
MS: tR = 2.6-2.8 min (3-40% B in 4 min); m/z = 807.8 ((M+2H)2+, calc.:
807.4), 538.9 ((M+3H)3+, calc.: 538.6); C70H112N22O20S: 1613.9 g·mol−1
.
2G-MAIP: For reductive amination, the peptidyl-resin (20 µmol) was
incubated with a mixture of ketone 10 and NaCNBH3 (15 eq each, c = 0.4
M) in DMF/MeOH/AcOH/TMOF (3/3/3/1) for 16 h. Semi-preparative HPLC:
3-45% B in 30 min; UPLC: tR = 2.7 min (3-40% B in 4 min); ESI-MS: 1103.6
((M+H)+, calc.: 1103.5), 552.4 ((M+2H)2+, calc.: 552.3); C47H70N14O15S:
1103.20 g·mol−1; yield: 3.97 µmol (20%; A280 = 0.99, V= 4 ml).
Synthesis of ligation products
Auxiliary peptides and peptide thioesters were united from stock solutions
in a 1:1 stoichiometry. The solution was lyophilized. The residue was
dissolved under argon atmosphere in degassed buffer (20 mM TCEP, 100
mM Na2HPO4, pH = 7.5, 3 vol% thiophenol) to final concentrations as
indicated. For isolation of ligation product, the mixture was separated by
semi-preparative HPLC and the united product fractions were lyophilized.
2G-MPE and 2N-MPE were synthesized as described recently.[12]
2G-ME and 2N-ME: For reductive amination, the peptidyl-resins were
incubated with a mixture of aldehyde 15 and NaCNBH3 (15 eq each relative
to resin-loading, c = 0.4 M) in NMP/iPrOH (3/1) with 5 vol% AcOH for 16
h. Semi-preparative HPLC: 3-40% B in 4 min. 2G-ME: Synthesis
scale:17.9 µmol; UPLC-MS: tR = 2.2 min; m/z = 968.6 ((M+H)+, calc.:
5GG-MAIP: Synthesis scale: 2.0 µmol; concentration: 4 mM; reaction time:
2 h, semi-preparative HPLC: 3-45% B in 30 min; UPLC-MS: tR = 2.87 min
(3-40% B in 2 min); 832.7 ((M+2H)2+, calc.: 832.4), 555.8 ((M+3H)3+, calc.:
555.4); C73H110N22O21S: 1663.85 g·mol−1; yield: 1.32 µmol (66%; A280
0.74, V = 2 ml).
=
968.5), 484.9 ((M+2H)2+, calc.: 484.7); C40H65N13O13S: 968.1 g·mol−1
;
yield: 3.18 µmol (18%; A280 = 0.408, V = 1.0 ml). 2N-ME: synthesis scale:
12.8 µmol; UPLC-MS: tR = 2.2 min (3-40% B in 4 min); m/z = 1025.6
((M+H)+, calc.: 1025.5), 513.3 ((M+2H)2+, calc.: 513.2); C42H68N14O14S:
1025.2 g·mol−1; yield: 4.91 µmol (38%; A280 = 0.628, V = 1.0 ml).
5GG-MAP: Synthesis scale: 2.3 µmol; concentration: 3 mM; reaction time:
3 h; semi-preparative HPLC: 3-45% B in 30 min; UPLC: tR = 3.12 min (3-
40% B in 2 min); ESI-MS: 833.1 ((M+2H)2+, calc.: 832.4), 555.5 ((M+3H)3+
,
calc.: 555.4); C73H110N22O21S: 1663.85 g·mol−1; yield: 1.12 µmol (56%;
280 = 0.23, V = 5.5 ml).
2G-MAP: The peptide resin (6.3 µmol) was treated with a mixture of the
MAP-auxiliary glycine conjugate 19 (6 eq, c = 0.4 M in DMF), PyBOP (6
eq) and DIPEA (18 eq) for 1 h. UPLC: tR = 1.78 min (3-40% B in 2 min);
ESI-MS: 1103.5 ((M+H)+, calc.: 1103.5), 552.4 ((M+2H)2+, calc.: 552.3);
C47H70N14O15S: 1103.20 g·mol−1; 1.4 µmol (22%; A280 = 0.69, V = 2 ml).
A
5GG-MPE: see reference[12]
5GG-ME: Synthesis scale: 1.0 µmol; concentration: 2 mM; reaction time:
1.5 h; semi-preparative HPLC: 3-40% B in 30 min; UPLC-MS: tR = 2.5 min
Peptide ligations
(3-40% B in 4 min); m/z = 765.1 ((M+2H)2+, calc.: 764.9), 510.7 ((M+3H)3+
,
calc.: 510.3); C66H105N21O19S: 1528.8 g·mol−1; yield: 0.73 µmol (73%; A280
= 0.373, V = 0.5 ml).
Auxiliary peptide 2X-Aux (1 eq) and peptide thioester 1Z (1-1.1 eq) were
united (from stock solutions) and lyophilized. The lyophilized peptides
were dissolved under argon atmosphere in degassed ligation buffer (20
mM TCEP, 100 mM Na2HPO4, pH = 7.5, 3 vol% thiophenol) to a final
concentration of 2-5 mM (details see SI). The progress of the reaction was
monitored by UPLC and HPLC-MS analysis. Peak areas were determined
with due regard to the molar extinction coefficient of the peptides.
5GG-MP: Synthesis scale: 1.0 µmol; concentration: 2 mM; reaction time:
4 h; semi-preparative HPLC: 3-40% B in 30 min; UPLC-MS: tR = 2.6 min
(3-40% B in 4 min); m/z = 772.3 ((M+2H)2+, calc.: 771.9), 515.2 ((M+3H)3+
,
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10.1002/chem.201705927
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calc.: 514.9); C67H107N21O19S: 1542.8 g·mol−1; yield: 0.63 µmol (63%; A280
= A280 = 0.323, V = 0.5 ml).
Keywords: peptide ligation • radical reactions • protecting
groups • peptide coupling • fragmentation
NMR-experiments
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a) P. Dawson, T. Muir, I. Clark-Lewis, S. Kent, Science 1994, 266, 776-
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K. S. A. Kumar, S. N. Bavikar, L. Spasser, T. Moyal, S. Ohayon, A. Brik,
Angew. Chem. Int. Ed. 2011, 50, 6137-6141.
Cleavage from 13C-labeled ligation product 5GG-MPE*: The lyophilized
13C-labbeled ligation product S31 (300 nmol, for synthesis see SI) was
dissolved in of a solution of 200 mM TCEP and 800 mM morpholine in a
mixture of (H2O/D2O, 95/5, v/v) to a final concentration of 0.5 mM. The
mixture was agitated at rt. After 24 h the aqueous phase was extracted
with deuterated chloroform (3 x 200 µL) and the organic phase was dried
over MgSO4. The aqueous and organic phase were analysed by UPLC-
MS and NMR spectroscopy. Please consult the SI (Table S1) for relevant
signals of the 1H-13C-HSQC analysis, detailed experimental setup and
preparation of reference materials. Cleavage from unlabeled 5GG-MPE:
The lyophilized ligation product 5GG-MPE (350 nmol, for synthesis see ref
[12]) was dissolved in a solution of 200 mM TCEP and 800 mM morpholine
in deuterated water to a final concentration of 0.5 mM. The mixture was
agitated at 40 °C. After 16 h the aqueous phase was extracted with
deuterated chloroform (3 x 200 µL) and the organic phase was dried over
MgSO4. The aqueous and organic phase were analysed by UPLC-MS and
NMR spectroscopy (Fig. S20).
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Becker, Angew. Chem. Int. Ed. 2015, 54, 7711-7715; d) C. Nadler, A.
Nadler, C. Hansen, U. Diederichsen, Eur. J. Org. Chem. 2015, 2015,
3095-3102.
Auxiliary cleavage
[9]
L. Z. Yan, P. E. Dawson, J. Am. Chem. Soc. 2001, 123, 526-533.
[10] Q. Wan, S. J. Danishefsky, Angew. Chem. Int. Ed. 2007, 46, 9248-9252.
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J. Chen, Q. Wan, Y. Yuan, J. Zhu, S. J. Danishefsky, Angew. Chem. Int.
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8090-8094; e) R. Yang, K. K. Pasunooti, F. Li, X.-W. Liu, C.-F. Liu, J. Am.
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Wan, S. J. Danishefsky, Tetrahedron 2010, 66, 2277-2283; g) Z. Harpaz,
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h) Z. Tan, S. Shang, S. J. Danishefsky, Angew. Chem. Int. Ed. 2010, 49,
9500-9503; i) S. Shang, Z. Tan, S. Dong, S. J. Danishefsky, J. Am. Chem.
Soc. 2011, 133, 10784-10786; j) P. Siman, S. V. Karthikeyan, A. Brik,
Org. Lett. 2012, 14, 1520-1523; k) L. R. Malins, K. M. Cergol, R. J. Payne,
ChemBioChem 2013, 14, 559-563; l) R. E. Thompson, B. Chan, L.
Radom, K. A. Jolliffe, R. J. Payne, Angew. Chem. Int. Ed. 2013, 52, 9723-
9727; m) L. R. Malins, K. M. Cergol, R. J. Payne, Chem. Sci. 2014, 5,
260-266; n) L. R. Malins, R. J. Payne, Aust. J. Chem. 2015, 68, 521-537;
o) J. Sayers, R. E. Thompson, K. J. Perry, L. R. Malins, R. J. Payne, Org.
Lett. 2015, 17, 4902-4905.
5GG-MAIP→4GG: The lyophilized ligation product 55G-MAIP (177 nmol)
was dissolved in 354 µL of a solution of 20 mM TCEP and 140 mM
morpholine in water. The mixture was agitated at 40 °C. After 22h, 93% of
the mixture was submitted to purification by semi-preparative HPLC
followed by lyophilization. 4GG: UPLC: tR = 1.15 min (3-40% B in 2 min);
ESI-MS: 1469.6 ((M+H)+, calc.: 1468.8), 735.0 ((M+2H)2+, calc.: 734.9);
490.5 ((M+3H)3+, calc.: 490.3); C64H101N21O19: 1468.61 g·mol−1; yield: 82
nmol, 50%; A280 = 0.165, VProbe = 500 µL.
5GG-MAP→4GG: The lyophilized ligation product 55G-MAP (200 nmol)
was dissolved in 400 µL of a solution of 20 mM TCEP and 140 mM
morpholine in water. The mixture was agitated at 40 °C. After 16 h, 93%
of the mixture was submitted to purification by semi-preparative HPLC
followed by lyophilization. 4GG: 83 nmol, 45% yield.
5GG-ME→4GG: The lyophilized ligation product 55G-ME was dissolved
in a solution of 100 mM TCEP and 400 mM morpholine in water to a final
concentration of 0.5 mM. The mixture was agitated at 40 °C. The progress
of the reaction was monitored by UPLC-MS analysis. 4GG: UPLC-MS: tR
= 2.4 min (3-40% B in 4 min); m/z = 735.1 ((M+2H)2+, calc.: 734.9), 490.5
((M+3H)3+, calc.: 490.3); C64H101N21O19: 1468.6 g·mol−1.
[12] S. F. Loibl, Z. Harpaz, O. Seitz, Angew. Chem. Int. Ed. 2015, 54, 15055-
15059.
[13] J. Vizzavona, F. Dick, T. Vorherr, Bioorg. Med. Chem. Lett. 2002, 12,
1963-1965.
5GG-MP→4GG: The lyophilized ligation product S29GG was dissolved in
a solution of 100 mM TCEP and 400 mM morpholine in water to a final
concentration of 0.5 mM. The mixture was agitated at 40 °C. The progress
of the reaction was monitored by UPLC-MS analysis. 4GG: UPLC-MS: tR
= 2.4 min (3-40% B in 4 min); m/z = 735.1 ((M+2H)2+, calc.: 734.9), 490.5
((M+3H)3+, calc.: 490.3); C64H101N21O19: 1468.6 g·mol−1.
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Acknowledgements
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2009, 10, 614-625; b) G. Rajpal, P. Arvan, in Handbook of Biologically
Active Peptides (Second Edition), Academic Press, Boston, 2013, pp.
1721-1729.
The authors gratefully acknowledge the financial support by the
Deutsche Forschungsgemeinschaft (Se819-15/1, SPP 1623).
[20] C. Walling, R. Rabinowitz, J. Am. Chem. Soc. 1959, 81, 1243-1249.
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10.1002/chem.201705927
Chemistry - A European Journal
FULL PAPER
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6759.
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Entry for the Table of Contents (Please choose one layout)
FULL PAPER
Simon F. Loibl, Andre Dallmann,
O
O
R
R
R
Kathleen Hennig, Carmen Juds and
Oliver Seitz*
Peptide
Peptide
Peptide
HS
Peptide
Peptide
N
N
HN
Aux
HS
H
O
O
O
Aux
HS
<
<
<
HS
HS
Reactivity:
Ar
HS
Page No. – Page No.
Ar/CH3
Ar
Features of Auxiliaries that Enable
Native Chemical Ligation beyond
Glycine and Cleavage via Radical
Fragmentation
Efficient peptide ligation auxiliaries have a β-mercaptoethyl scaffold and avoid
substituent in α–position to the amine. Surprisingly, β-substituents accelerate
ligation reactions. All presented auxiliaries can be removed under mild-basic
conditions. NMR analysis of a 13C-labelled auxiliary indicated that auxiliary
cleavage most likely proceeds through a radical oxidation-fragmentation cascade.
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