Selective Bi-directional amide bond cleavage of N-methylcysteinyl peptide

Article abstract of DOI:10.1002/ejoc.201402261

A selective bi-directional peptide bond cleavage mediated by N-methylcysteine (MeCys) in Xaa-MeCys-Yaa peptides (Xaa and Yaa, non-cysteine residues) leading to thioesters and thiolactones is described. Rate and product analyses showed that an N^α-amide bond cleavage occurred at the Xaa-MeCys bond by an N-S acyl shift to generate an Xaa-S-(MeCys-Yaa) thioester at pH 1-5, whereas under strongly acidic conditions of H₀ = -5, the MeCys-Yaa bond underwent a C^α-amide bond cleavage via an oxazolone intermediate, which was trapped by thiocresol (TC) as an Xaa-MeCys-TC thioester. This thioester was then transformed into an Xaa-MeCys-β- thiolactone at pH 4-5. Replacing MeCys by a Cys residue did not result in significant bi-directional peptide bond cleavage, which suggests that N-methylation in a MeCys residue is important for the N-S acyl shift reaction and formation of oxazolone. The isomerization of amides and thioesters was successfully used to prepare cyclic peptides. Copyright

Full text of DOI:10.1002/ejoc.201402261

FULL PAPER
DOI: 10.1002/ejoc.201402261
Selective Bi-directional Amide Bond Cleavage of N-Methylcysteinyl Peptide
Yibo Qiu,[a] Xinya Hemu,[a] Ding Xiang Liu,[a] and James P. Tam*[a]
Keywords: Peptides / Amides / Cleavage reactions / Cyclization
A selective bi-directional peptide bond cleavage mediated
by N-methylcysteine (MeCys) in Xaa-MeCys-Yaa peptides
(Xaa and Yaa, non-cysteine residues) leading to thioesters
and thiolactones is described. Rate and product analyses
showed that an Nα-amide bond cleavage occurred at the
Xaa-MeCys bond by an N–S acyl shift to generate an
Xaa-S-(MeCys-Yaa) thioester at pH 1–5, whereas under
strongly acidic conditions of H0 = –5, the MeCys-Yaa bond
underwent a Cα-amide bond cleavage via an oxazolone in-
termediate, which was trapped by thiocresol (TC) as an Xaa-
MeCys-TC thioester. This thioester was then transformed
into an Xaa-MeCys-β-thiolactone at pH 4–5. Replacing
MeCys by a Cys residue did not result in significant bi-direc-
tional peptide bond cleavage, which suggests that N-methyl-
ation in a MeCys residue is important for the N–S acyl shift
reaction and formation of oxazolone. The isomerization of
amides and thioesters was successfully used to prepare cyclic
peptides.
Two general Fmoc-compatible approaches to the prepa-
ration of thioesters have been advanced. Both approaches
employ thioester surrogates that are resistant to organic
bases in deprotection steps during the peptide-chain-
assembly steps in Fmoc chemistry. However, they differ in
their post-chain-assembly step to generate a peptide thioes-
ter. The first approach, “safety catch”, can be characterized
by the prototypical thioester surrogate, sulfonamide.[6]
Sulfonamides are resistant to piperidine, serving as a
“safe” thioester surrogate in the deprotection steps in Fmoc
chemistry. However, they can be activated by sulfonamide
N-alkylation to yield an N-substituted sulfonamide and the
subsequent “catch” reaction using a thiol nucleophile in an
intermolecular SN2 substitution reaction leads to a thio-
ester, generally under basic conditions. Variations of the
“safety catch” approach include the hydrazide,[7] pyroglut-
amyl imide,[8] and N-acylurea methods.[9]
The second approach can be characterized as a “safety
switch” strategy. Esters, or more commonly, amides have
been used as thioester surrogates in this approach.[10] The
key reaction here is an isomerization of an amide to a thio-
ester by an N–S acyl shift reaction to afford a peptide thio-
ester. Differing from the “safety catch” methods, which in-
volve post-chain-assembly activation and intermolecular
thiol substitution, the formation of a thioester in the “safety
switch” approach occurs by an intramolecular reaction me-
diated by an N–S acyl shift and is generally acid-catalyzed.
The “safety switch” approaches to the preparation of a thio-
ester from an amide include methods involving a cysteinyl
propyl ester,[11] N-alkylcysteine,[12] thiazolidine,[10b] SEA-
anilide,[13] mercaptomethylated proline,[14] a bis(thioethyl)
amino group,[15] and recently the TEBA group.[16]
Introduction
Thioesters are versatile functional groups and are widely
used in biological and chemical reactions, particularly in
the breaking and forming of peptide bonds. For example,
in proteolysis, thioesters are intermediates of thiol proteases
in the breaking of peptide bonds[1] and in ligation reactions
of peptides and proteins, thioesters are key intermediates
that enable proximity-driven ligation for the formation of a
new peptide bond.[2] Ligation reactions are particularly use-
ful for preparing cyclic peptides through intramolecular lig-
ation of a linear precursor containing both an N-terminal
Cys residue and a C-terminal thioester.[3]
For the chemical synthesis of cyclic peptides, an efficient
and practical method for the preparation of a C-terminal
peptide thioester is necessary. Peptide thioesters can be con-
veniently obtained by tert-butoxycarbonyl (Boc) chemistry
in solid-phase synthesis,[4] but their synthesis remains a
challenge by 9-fluorenylmethoxycarbonyl (Fmoc) chemis-
try. In Fmoc chemistry, thioesters are susceptible to piper-
idine treatment, which is employed repetitively during de-
protection steps. However, Fmoc chemistry avoids the use
of strong acids such as HF in the final cleavage step and
is more suitable than Boc chemistry for the synthesis of
phosphorylated and glycosylated peptides, which are sensi-
tive to strong acids. Consequently, various methods have
been reported for the preparation of thioesters compatible
with Fmoc chemistry in solid-phase peptide synthesis.[5]
[a] School of Biological Sciences, Nanyang Technological
University,
60 Nanyang Drive, Singapore 637551, Singapore
E-mail: jptam@ntu.edu.sg
http://www.sbs.ntu.edu.sg/aboutus/Faculty/JPTam/Pages/
Home.aspx
The isomerization of an Xaa-Cys bond leads to a thioes-
ter from a cysteine amide bond and is the reverse process of
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402261.
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Amide Bond Cleavage of N-Methylcysteinyl Peptide
Scheme 1. Thioester-Cys ligation and the reverse thioester formation through an N–S acyl shift; R1 = alkyl or aryl groups, R2 = alkyl or
aryl groups assisting the N–S acyl shift.
Scheme 2. Bi-directional bond cleavage mediated by MeCys.
the native chemical ligation (Scheme 1). Common to both Nα-cleavage of the Xaa-MeCys bond to form an Xaa-thio-
reactions is the thioethylamido moiety that enables a prox- ester or a thiolactone inter- or intramolecularly by an N–S
imity-driven N–S or S–N acyl shift reaction via a five-mem- acyl shift and a thiol/thioester exchange reaction with the
bered-ring transition state to form a thioester or an amide, concomitant release of the MeCys residue. Under strongly
respectively.[16] Although the native chemical ligation pro- acidic conditions at Ho = –5, the ethyl thiol in the thioethyl-
cess is performed under basic conditions, amide-to-thioester amido moiety is strongly protonated, which diminishes its
reversal is conducted under acidic conditions. Additionally, ability to participate in the Nα-cleavage. Consequently, an
the N–S acyl shift to form a thioester from an amide is Xaa-MeCys-thioester was formed by Cα-cleavage of the
facilitated by the scissile peptide bond in a cis conforma- MeCys-Yaa bond without the loss of the MeCys residue.
tion, which mimics the enzymatic conformational assistance Cα-cleavage of the MeCys residue was found to proceed via
of the N–S acyl shift in protein splicing.[17] MeCys repre- an N-methyloxazolone intermediate, which is trapped by
sents a simple thioester surrogate that enables cleavage of a thiocresol as a thiocresol thioester. At pH 4–5, this thioester
ciscoid amide bond by an N–S acyl shift reaction leading was readily transformed by the thioethylamido moiety into
to a thioester under the “safety switch” approach.[16a] How- an Xaa-MeCys-β-thiolactone in a previously unreported re-
ever, N-alkyl amino acids are known to be susceptible to action, which could provide a route to the preparation of
acid-catalyzed racemization, a side-reaction likely involving the thioacid for peptide ligation.[18] MeCys serves as a thio-
oxazolone formation and an amide bond cleavage, but Cα- ester surrogate and is fully compatible with Fmoc chemistry
amide bond cleavage of MeCys at its carboxy side has not in solid-phase peptide synthesis, and was exploited to form
been studied. For convenience, in this work we define Nα- a thioester in the synthesis of sunflower trypsin inhibitor-1
and Cα-cleavage as Nα-amide and Cα-amide bond cleavage, (SFTI-1), a 14-residue cyclic peptide and the smallest and
respectively.
most potent peptidyl inhibitor in the Bowman–Birk class of
Herein we describe the use of the MeCys residue of an protease inhibitors.[19]
Xaa-MeCys-Yaa peptide in a bi-directional cleavage of
peptide bonds leading to the formation of a thioester or
thiolactone at either the N- or C-terminal side of the Me-
Cys residue by manipulating the acidity of the reaction con-
Results and Discussion
Synthetic Strategy
ditions (Scheme 2). Under aqueous acidic conditions at
Our strategy for the synthesis of a cyclic peptide em-
pH 1–5, the thioethylamido moiety in MeCys mediated an ployed a general design of “Cys-peptide-MeCys” as a linear
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Y. Qiu, X. Hemu, D. X. Liu, J. P. Tam
FULL PAPER
Scheme 3. Tandem thiol switch reaction for the formation of a thioester from the MeCys thioester.
precursor (1) with a Cys and MeCys residue flanking the chemistry to obtain TR6 (9a) and SFTI-1 (9b). TR6 (9a)
peptide of interest at its N- and C-termini (Scheme 3). Such with the linear sequence TIGGIR was synthesized to con-
a design should permit a one-pot reaction leading to a cy- duct a kinetic study of the N–S acyl shift and thiol/thioester
clic peptide from its linear precursor through two types of exchange
reactions.
SFTI-1,
cyclo-(G1RCTKSIP-
acyl shift reactions and a thiol/thioester exchange reaction. PICFPD14) with a disulfide bond between Cys3 and Cys11,
These are 1) an N–S acyl shift mediated by MeCys to form was chosen as a model to demonstrate the synthesis of cy-
a thioester or thiolactone 2, 2) thiol/thioester exchange be- clic peptides, highlighting the use of MeCys as a thioester
tween the N-terminal cysteinyl thiol and the C-terminal surrogate. Its precursor 9b was synthesized with Arg2-Cys3
thioester/thiolactone 3 mediated by Cys, and 3) an S–N acyl serving as the cyclization point.
shift to form macrolactam 4. Because the N–S acyl shift to
form a thioester is the reverse of the S–N acyl shift in the
intramolecular ligation reaction to form a cyclic peptide,
the proposed strategy exploits both the forward and reverse
acyl shift reactions to accomplish our goal of the synthesis
of the cyclic peptide sunflower trypsin inhibitor-1 (SFTI-1).
The commercially available Fmoc-MeCys(Trt)-OH was
used as the starting material and attached to the resin sup-
ports as a thioester surrogate in our stepwise Fmoc solid-
phase synthesis of SFTI-1. To provide insights into the
amide-to-thioester isomerization by an N–S acyl shift medi-
ated by MeCys, we also prepared model peptide TIGGIR-
Xaa-MeCys-Yaa to examine the effect of acidity on the for-
mation of a thioester.
Scheme 4. Synthesis of the MeCys peptide precursors 9a and 9b.
Synthesis of MeCys(Trt)-Resin and Model Peptides
To obtain a Cys-peptide-MeCys precursor, our strategy
In our previous synthesis of SFTI-1, we observed aspart-
started with the Rink amide resin (Scheme 4). Fmoc-Gly- imide formation at Asp14-Gly1 of SFTI-1. To minimize this
OH was coupled to the Rink resin by using N,NЈ-diisoprop- side-reaction, we modified the microwave-assisted Fmoc de-
ylcarbodiimide (DIC) and 1-hydroxy-7-azabenzotriazole protection protocol and used 20% morpholine in DMF
(HOAt) to generate resin 5 with a Gly spacer to prevent (3ϫ 5 min)[20] instead of the conventionally used 20% pi-
diketopiperazine formation. Following the removal of the peridine. In this synthesis of SFTI-1 on MeCys(Trt)-con-
Fmoc group by using 20% piperidine, Fmoc-N-methyl- taining resins, we observed another side-reaction, the for-
Cys(Trt) was coupled to the resin 5 by using (benzotriazol- mation of piperidinyl alanine, MeAla(Pip), a side-product
1-yloxy)tripyrrolidinophosphonium hexafluorophosphate with a mass 51 Da greater than the expected mass of Me-
(PyBOP) and N,N-diisopropylethylamine (DIEA). Because Cys(Trt). MeAla(Pip) was formed by base-catalyzed β-eli-
of the low reactivity of the secondary amine in MeCys(Trt), mination of a C-terminal MeCys(Trt) followed by the
the coupling reaction between MeCys(Trt)-Gly-resin and Michael addition of piperidine to form 3-(piperidin-3-yl)-
Fmoc-Arg(Pbf) to produce resin 7 was difficult and re- alanine during the Fmoc synthesis of a peptide containing
quired the use of O-(7-azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tet- a C-terminal MeCys(Trt) on the resin. This side-reaction of
ramethyluronium hexafluorophosphate (HATU) and three a C-terminal Cys(Trt) or Cys(Acm) attached directly to a
coupling reactions for completion. This process was moni- resin support was previously reported by Lukszo et al. in
tored by means of the acetaldehyde/chloranil test. Resin 7 1996,[21] but could be minimized by using a spacer. We
was subjected to automated peptide synthesis using Fmoc found that the MeAla(Pip) side-product accumulated at a
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Amide Bond Cleavage of N-Methylcysteinyl Peptide
rate of 0.3% per deprotection cycle in our Fmoc solid-phase was formed by thiol/thioester exchange of thioester 10a by
synthesis. The addition of a Gly as a spacer did not resolve using sodium 2-sulfanylethanesulfonate (MESNa; Fig-
the problem completely, which suggests that the common ure 1). As shown in Figure 1, the optimal pH range for the
occurrence of the β-elimination side-reaction when the Me- tandem thiol switch reaction was found to be pH 2–5. The
Cys(Trt) residue is proximally linked through the C ter- MES-thioester 11a was obtained in Ͼ90% yield at pH 2–5
minus to resin supports.
with the best yield obtained at pH 2, which afforded 95%
11a at 40 °C. At pHϽ2, the dominant presence of the thio-
ester (S form) 10a indicated that the N–S acyl shift was
favored over the thiol/thioester exchange reaction. At
pHϾ5, the starting material 9a predominated, which sug-
gests that the N–S acyl shift reaction was not preferred un-
Nα-Cleavage of MeCys in Xaa-MeCys-Yaa Peptides in
Aqueous Acidic Solutions
The Ser/Thr/Cys bonds in certain peptide sequences are der neutral or basic conditions. Side-reactions also occurred
known to be weak links and readily hydrolyzed under aque- at either low (pH 1) or high pH (pH 6–7). At pH 1, 10c, a
ous acidic conditions or susceptible to rearrangement under deamidation form of 10a at the C-terminal Gly residue, was
strongly acidic conditions.[3b] Interestingly, such bonds are observed. At pH 6 and 7, TIGGIR-OH, the hydrolysis
also used as cleavage sites by the enzyme intein during pro- product of the MES thioester, was detected. The synthetic
tein splicing through a series of acyl shifts involving an MES thioester 11a was used in a ligation reaction with N-
N–S or N–O acyl shift, an O–O or S–S exchange reaction, terminal cysteinyl peptide CALVIN to form TIGGIRCAL-
and then an uncatalyzed O–N or S–N acyl shift to join the VIN (see Figures S10 and S11 in the Supporting Infor-
extein fragments.[1a,1b] The Nα-cleavage of a Gly-Cys bond mation).
in trifluoroacetic acid (TFA) was studied in detail by Aim-
oto and co-workers.[22] They found that the cleavage of the
Gly-Cys bond in TFA undergoes an isomerization reaction
by an N–S acyl shift to produce both the N form (amide)
and S form (thioester). This isomerization is reversible and
slow at room temperature with a rate of 0.09% per hour.
However, the reversibility could be minimized by trapping
the S form as another thioester by performing two reactions
in tandem.[11] First, adding a Pro ester to the C terminus of
the Gly-Cys dipeptide to form a Gly-Cys-Pro ester enables
its S form to produce a diketopiperazine (DKP) as a Gly-
S-(DKP) thioester, which eliminates the Nα-amine of Cys
in the S–N acyl shift reaction. Secondly, adding a thiol to
the reaction mixture enables an exchange reaction of the
DKP thioester with an external thiol to form a stable thio-
ester through an thiol/thioester exchange reaction. This
method, the cysteinyl prolyl ester (CPE) approach to pre-
pare a thioester, has been extended by others to prepare a
thioester from bacterially expressed proteins by using the
lability of the Gly-Cys bond.[23] Hojo and co-workers also
developed a series of Cys derivatives by using Xaa-N-alkyl-
Cys (RCys, R = ethyl, propyl, and butyl) as a thioester sur-
rogate and a tandem thiol switch reaction by exploiting the
isomerization of the Xaa-RCys amide bond (N form) to its
thioester (S form), followed by a thiol/thioester exchange to
form another thioester under aqueous conditions using 3-
mercaptopropionic acid (MPA) or 4-mercaptophenylacetic
acid (MPAA).[12a,12b] However, a kinetic study of the Xaa-
MeCys bond cleavage and comparison of the acid lability
Figure 1. Tandem thiol switch reaction of TIGGIR-MeCys-Gly-
of Gly-Cys and Gly-MeCys bonds have not been reported,
NH2 after 24 h incubation at 40 °C in acidic aqueous solution with
and so to gain a greater understanding of the Nα-cleavage
50 equiv. MESNa (buffer B gradient: 10–60% ACN in 30 min,
room temperature).
and tandem thiol switch reactions we used our model pept-
ide TIGGIR (TR6).
The model peptide TR6-MeCys-Gly-NH2 (9a) was used
To investigate the effects of N-methylation of a Cys resi-
to determine the effect of acidity on the tandem thiol switch due on the Nα-cleavage of the Gly–Cys or Gly–MeCys
reaction involving the N–S acyl shift and thiol/thioester ex- bond, two model peptides, TR6-Gly-Cys-Gly-NH2 and
change reactions. The model peptide (N form) 9a is in equi- TR6-Gly-MeCys-Gly-NH2, were synthesized. For the tan-
librium with the thioester (S form) 10a. MES thioester 11a dem thiol switch reaction, 20% methyl mercaptoacetate
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Y. Qiu, X. Hemu, D. X. Liu, J. P. Tam
FULL PAPER
(MMA) instead of 50 equiv. MESNa was chosen as the ex-
Thus, we anticipated that the tandem thiol switch reac-
ternal thiol additive for two reasons. First, the MES-thioes- tions mediated by MeCys under strongly acidic conditions
ter and its precursor TR6-Gly-Cys-Gly-NH2 were poorly would also be accelerated. Unexpectedly, treatment of TR6-
separated by HPLC. Secondly, MMA is a stronger nucleo- MeCys-Gly-NH2 (9a) with 0.1% TFMSA and 5% TC in
phile to facilitate the thioesterification of the Gly–Cys bond TFA for 2 h gave Ͻ5% of the desired product TR6-TC by
than MESNa. Because the external thiol was used in a large Nα-cleavage of the Arg-MeCys bond, as determined by RP-
excess for the thiol/thioester exchange reaction, the overall HPLC. Instead, Cα-cleavage of the MeCys-Gly bond oc-
rate of the tandem thiol switch reaction can be calculated curred and TR6-MeCys-TC was obtained as the major
as a first-order reaction. The yield of the thioester TR6- product (75%). TR6-MeCys-TC was unambiguously con-
Gly-MMA was determined by RP-HPLC (Figure 2). Based firmed by MALDI-TOF tandem mass spectrometry (MS/
on the initial rates, MeCys mediates the tandem thiol switch MS), with each fragmented mass of TIGGIR-MeCys-TC
reaction 150-fold faster than Cys, that is, 0.31 min–1 for Me- clearly identified (see Figure S12 in the Supporting Infor-
Cys and 0.002 min–1 for Cys. Under our experimental con- mation). In aqueous buffer containing 0.1% TFA, TIG-
ditions, the Gly-Cys bond is very stable and significant GIR-MeCys-TC was observed to form an equilibrium be-
isomerization between amide and thioester forms will not tween amide 15 (N form) and thioester 15Ј (S form, Fig-
occur whereas the Gly-MeCys bond completed the trans- ure 3). This result is consistent with our work described
formation from an amide to thioester within 9 h. These re- above, which showed that peptides containing MeCys are
sults clearly show that N-methylation is important for exert- in equilibrium between the amide and thioester forms due
ing conformational effects in the N–S acyl shift reaction. to the N–S acyl shift reaction of the MeCys residue under
They also suggest that our synthetic scheme is compatible aqueous acidic conditions[12a] and further confirmed that
with cysteine-rich peptides without any fragmentation TIGGIR-MeCys-TC (15) contains the MeCys residue. Ad-
problems of internal cysteine junctions under our experi- ditionally, TR6-MeCys-TC (15) was readily ligated with the
mental conditions.
cysteinyl peptide CALVIN to produce TIGGIR-MeCys-
CALVIN under the standard ligation conditions at pH 7.5.
The ligation product was confirmed by MS/MS (see Fig-
ures S13 and S14 in the Supporting Information).
Figure 2. Comparison of Cys- and MeCys-mediated Nα-cleavage of
a Gly-MeCys or Gly-Cys bond. TIGGIR-Gly-MeCys-Gly-NH2
and TR6-Gly-Cys-Gly-NH2 were treated with 20% MMA (v/v) at
pH 2 at 40 °C.
Cα-Cleavage of MeCys in Xaa-MeCys-Yaa Peptides under
Strongly Acidic Solutions
Figure 3. RP-HPLC traces of TR6-MeCys-TC in 0.1% TFA in
H2O after 0 and 12 h. N form: TR6-MeCys-TC amide form; S
form: TR6-MeCys-TC thioester form.
Previously, we reported the use of thioethanol and 2-
thiomethylthiazolidine as Fmoc-compatible thioester surro-
Cα-cleavage of the MeCys-Gly bond leading to the for-
gates,[10a,10b] which can be transformed into thioesters by mation of TR6-MeCys-TC from TR6-MeCys-Gly-NH2,
an acid-catalyzed tandem thiol switch reaction using 0.1% which retains the MeCys residue, is unusual as peptide
trifluoromethanesulfonic acid (TFMSA) in TFA and thio- bonds are generally stable to strong acids, but sequence-
cresol (TC). Thiocresol was selected as a thiol nucleophile dependent cleavage reactions are known and are often side-
because it is a weaker base than alkyl thiols, which do not chain-assisted. To determine whether the Cα-cleavage of
work well under strongly acidic conditions. This one-pot MeCys is sequence-dependent, TR6-Xaa-MeCys-Yaa-NH2
reaction completed within 2 h is considerably faster than peptides (Xaa and Yaa are different amino acids) were
the corresponding reaction performed under aqueous acidic treated with 0.1% TFMSA in TFA. The product profile
conditions, producing a thioester with concurrent side- is summarized in Table 1. TR6-Xaa-MeCys-TC, TR6-Xaa-
chain deprotection and cleavage from resin supports.
MeCys-OH, and their S forms, which were likely formed
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Amide Bond Cleavage of N-Methylcysteinyl Peptide
Table 1. Product analysis of TIGGIR-Xaa-MeCys-Yaa treated with 0.1% TFMSA and 5% thiocresol.
Type of
cleavage
Product
Yield [%]
Xaa
Yaa
[a]
–
Gly
Ala
Leu
NH2
Gly[b]
Ala[b]
Nα-Cleavage
Cα-Cleavage
TR6-Xaa-MeCys-Yaa (S form)
TR6-Xaa-TC
Total
TR6-Xaa-MeCys-OH (S form)
TR6-Xaa-MeCys-OH (N form)
TR6-Xaa-MeCys-TC (S form)
TR6-Xaa-MeCys-TC (N form)
Total
1.6
1.4
3.0
7.7
10.4
3.9
4.8
1.1
5.9
5.0
8.4
3.5
77.2
94.1
0.5
1.8
2.3
2.6
16.7
8.9
1.0
1.0
2.0
3.6
14.0
6.0
5.3
5.4
10.7
18.0
17.9
6.1
1.6
1.4
3.0
7.7
10.4
3.9
5.2
2.5
7.7
28.5
13.2
2.2
48.4
92.3
75.1
97.0
69.4
97.7
74.3
98.0
47.4
89.3
75.1
97.0
[a] – refers to no amino acid between TR6 and MeCys. [b] Gly and Ala refer to Gly-NH2 and Ala-NH2 respectively.
during RP-HPLC purification at pH 2, were derived from Ͻ1% yield in 2 h and Ͻ10% yield in 170 h when the Cys
the Cα-cleavage reaction of the MeCys residue. The remain- peptide was used.
ing two products were TR6-TC and the TR6-MeCys-Yaa-
To determine the acidity function of 0.1% TFMSA in
NH2 peptide S form, which result from Nα-cleavage of the TFA, we used 1H NMR spectroscopy to measure the chem-
MeCys residue. Our results show that Cα-cleavage of Me- ical shifts of the protonated and unprotonated forms of di-
Cys is not substantially affected by the nature of the amino methyl sulfide (DMS) in various concentrations of TFMSA
acids at either side of the MeCys residue under strongly in TFA.[24] The results of the NMR titrations show that the
acidic conditions.
acidic function Ho of the 0.1% TFMSA solution in TFA is
When a different Yaa amino acid (Ala, Gly, or NH2) was –5. Thus, there is a log scale difference of seven between the
used, TR6-MeCys-TC was invariably formed irrespective of acidity of an Nα-cleavage, which is optimal at pH 2, and a
the C-terminal amino acid (Table 1, right column). From Cα-cleavage, which is strongly favored under strongly acidic
Ala to Gly, a comparable Cα-cleavage yield was obtained conditions with Ho = –5.
(89.3–97.0%). These results show that the Cα-cleavage reac-
Thus far, our results show that the Cα-cleavage reaction
tion was not restricted to the presence of Gly in the TR6- of MeCys under strongly acidic conditions is not sequence-
MeCys-Gly-NH2 peptide. To study the sequence-dependent dependent as the amino acid at its N- or C-terminal side
effect of the N-terminal amino acid of MeCys in the Cα- exerts little effect on the formation of the C-terminal thio-
cleavage of a MeCys-Xaa bond, we synthesized four pept- cresol thioester. Furthermore, replacing MeCys by MeAla
ides, TR6-Xaa-MeCys-Gly-NH2 with Arg, Gly, Ala, and resulted in the formation of the C-terminal thiocresol thio-
Leu at the N-terminal MeCys (Table 1, left column). Gly ester. However, when the N-methyl group of MeCys was
was chosen as the C-terminal amino acid because it gave removed and replaced by Cys in the Xaa-MeCys-Yaa pept-
the highest yield. No substantial difference was observed in ides, the Cα-cleavage reaction was strongly suppressed,
the Cα-cleavage yield by using different amino acids at the which demonstrates the importance of the N-methyl group
N-terminal side of MeCys, with yields ranging from 94– in this reaction. N-Alkyl amino acids are known to be sus-
98%. The presence of a basic residue Arg in our model ceptible to racemization in strong acids such as HBr in ace-
peptide TR6 did not significantly affect the Cα-cleavage of tic acid,[25] and oxazolone formation has long been sus-
TR6-MeCys-Gly-NH2, leading to a thioester as well as its pected as the cause. Because the acidity of 0.1% TFMSA
corresponding peptide with an alkyl amino acid such as Ala in TFA (Ho = –5) is comparable to HBr in acetic acid, ox-
and Leu.
azolone formation is likely the mechanism in our observed
To determine whether the Cα-cleavage reaction is assisted Cα-cleavage reaction.
by the thioethyl side-chain of MeCys, we prepared TR6-
Scheme 5 shows the proposed mechanism for the Cα-
MeAla-Gly, replacing MeCys with MeAla. Treatment of cleavage reaction via an N-methyloxazolone intermediate
TR6-MeAla-Gly with 0.1% TFMSA and 5% TC produced leading to the formation of thioester TR6-MeCys-TC or
TR6-MeAla-TC in 75% yield, a result similar to treating TR6-MeAla-TC. The formation of oxazolone from an N-
TR6-MeCys-Gly-NH2 under the same conditions. Taken methylamino acid residue involves both carbonyl groups lo-
together, these results suggest that the N-alkylation of an cated at the N- and C-terminal sides. When the N-methyl
amino acid strongly accelerates the Cα-cleavage reaction un- amide bond was strongly protonated, an N-methyloxazol-
der strongly acidic conditions. This observation was further one was produced by nucleophilic attack of the N-terminal
confirmed by replacing MeCys by Cys in the model peptide carbonyl oxygen on the C-terminal carbonyl side of the N-
TR6-Gly-Xaa-Gly-NH2, which did not result in significant methylamino acid. In our case, the reactive N-methyloxaz-
Cα-cleavage. The model peptides TR6-Gly-Cys-Gly-NH2 olone intermediate would be trapped by a large excess of
and TR6-Gly-MeCys-Gly-NH2 were each treated with thiocresol (TC) to form the thioester. The proposed mecha-
0.1% TFMSA and 5% TC in TFA. Treatment of the Me- nism is consistent with our data and is supported by Ur-
Cys peptide for 1 h resulted in 75% yield of the thioester. ban[26] and Teixido[27] and their co-workers who reported
In contrast, the product TR6-Gly-Cys-TC was obtained in that peptides containing N-alkylated amino acids in TFA
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Scheme 5. Proposed mechanism for N-methylamino acid-Yaa bond cleavage.
for a prolonged period in resin or the solution phase are displacement reaction, would involve a four-membered-ring
prone to cleavage at the C-terminal side of an N-alkylated intermediate leading to β-thiolactone formation. Indeed,
amino acid to form a hydrolysis product. The N-alkylation treatment of TR6-MeCys-TC in aqueous buffer solutions
of an amino acid residue plays a key role in the activation at pH 5 and 6 generated TR6-MeCys β-thiolactone (m/z =
of the amide bond on the N-terminal carbonyl side, which 715; Scheme 6). The conversion of TR6-MeCys-TC into its
induces nucleophilic attack on the C-terminal carbonyl side. β-thiolactone was rapid, as shown by RP-HPLC analysis
Thus, under strongly acidic conditions, when the ethyl thiol (Figure 4, A,B).
of the thioethylamido moiety is strongly protonated thereby
reducing its nucleophilicity, Nα-cleavage is suppressed and
Cα-cleavage predominates.
The addition of TFMSA favored Cα-bond cleavage in the
Xaa-MeCys-Yaa peptide and significantly increased the for-
mation of oxazolone. Under our conditions using 0.1%
TFMSA, all Cα-cleavage reactions were completed within
1 h, whereas the acid hydrolysis reaction of MeAla, MeGly,
or MePhe in TFA (H0 = –3) reported by Urban and co-
workers required 13.5–64.5 h for completion.[26] The use of
a very weak base such as thiocresol (TC) could also con-
tribute to the acceleration of the reaction rate because TC
Scheme 6. Formation of β-thiolactone.
is a better nucleophile than H2O, which leads to acid hy-
To show the importance of the thioethyl side-chain of
drolysis of N-methylamino acids. It should be pointed out
MeCys in forming the β-thiolactone, the thiol moiety of the
that MeCys was not studied by Urban and co-workers.
MeCys residue was S-alkylated by N-ethylmaleimide
(NEM). The S-alkylated TR6-MeCys-TC was stable in
aqueous solutions at pH 5, with no β-thiolactone formation
observed at 12 h (Figure 5). This clearly shows that the thiol
side-chain of MeCys is responsible for β-thiolactone forma-
tion. The TR6-MeCys β-thiolactone has the same molec-
ular weight (m/z = 715) as the lactones, lactams, or end-to-
end macrocycles formed by the Thr side-chain, the Arg
We also noted that the formation of N-methyloxazolone
was not observed in the 2-thiomethylthiazolidine surrogates
when using 0.1% TFMSA in TFA. The formation of N-
methyloxazolone is not favored because the steric hindrance
of the thiazolidine ring impedes the oxygen from attacking
the C-terminal carbonyl to form the bicyclic oxazolone in-
termediate. This is consistent with our observation that re-
placing the MeCys residue with Pro, a native secondary
side-chain, or the N-terminal α-amine, respectively. How-
ever, we ruled out these possibilities based on the MS/MS
amino acid, did not result in any Cα-cleavage.
fragmentation pattern (see Figure S15 in the Supporting In-
formation) and the result of the S-alkylation. Replacing
MeCys by Cys also resulted in β-thiolactone formation un-
β-Thiolactone Formation
We sought to determine whether the thioethyl side-chain der similar conditions.
of the MeCys residue could mediate a proximity-driven Cα-
However, when methyl mercaptoacetate (MMA) thioes-
cleavage reaction when a good leaving group such as a C- ter was used instead of the TC thioester, no β-thiolactone
terminal TC thioester is present. Such a cleavage, or rather was obtained. Both TR6-MeCys-MMA and TR6-Cys-
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Amide Bond Cleavage of N-Methylcysteinyl Peptide
Our result is consistent with that of Johnson and Kent,[28]
who reported that aryl thioesters are more reactive than
alkyl thioesters in native chemical ligation.
Synthesis of SFTI
We synthesized SFTI-1 to illustrate our scheme, high-
lighting MeCys as a thioester surrogate through Nα-cleav-
age. Previously, the cyclized backbone of SFTI was pre-
pared by Boc/Fmoc chemical synthesis[29] and a recombi-
nant approach.[30] In the synthesis of SFTI, we envisioned
that the thiol/thioester exchange reaction would likely be
intramolecular, forming a thiolactone with one of the two
cysteinyl residues present in SFTI. Such a thiolactone is
expected to undergo another intramolecular S–N acyl shift,
which is common in thioester-Cys ligation, to form the cy-
clic SFTI under basic conditions. We previously reported
the sequential thiolactone and lactam formation as a thia
zip cyclization, and evidence for the formation of five of the
six possible thiolactones was observed during the cycliza-
tion of the cysteine-rich peptides cyclic conotoxin and kB1,
which contain six Cys moieties.[16a,31] Thus, the thia zip cy-
clization facilitates the synthesis of SFTI in a one-pot reac-
tion using an unprotected peptide thioester precursor.
The acid-catalyzed N–S acyl shift of SFTI-MeCys-Gly-
NH2 (9b) to give its MES thioester 11b was performed in
aqueous solution at pH 2, shown to be optimal for a tan-
dem thiol switch, as described in the previous section. An
Figure 4. Stability of TR6-MeCys-TC in pH 4–7 phosphate buffer.
A) pH 4, B) pH 5, C) pH 6, D) pH 7. 15: TR6-MeCys-TC, 16:
TR6-MeCys β-thiolactone, 17: TR6-MeCys-OH, 18: TR6-MeCys
diacyl-thioester.
Figure 5. Stability of NEM alkylated TR6-MeCys-TC in pH 5
phosphate buffer.
MMA were sufficiently stable in phosphate buffer at pH 5
up to 12 h, as revealed by RP-HPLC analysis. TIGGIR-
MeCys-TC, or more likely its β-thiolactone product, was
not stable at pH 6 and 7 and resulted in degraded products
after treatment with phosphate buffer at pH 6 or 7 for 1 h
(Figure 4, C,D). A plausible explanation is that TC, an aryl
Figure 6. Synthesis of cyclic sunflower trypsin inhibitor SFTI-1 by
a tandem thiol switch and thia zip cyclization. Each synthetic inter-
thiol, is a better leaving group than MMA, an alkyl thiol. mediate was purified and analyzed by RP-HPLC.
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Y. Qiu, X. Hemu, D. X. Liu, J. P. Tam
FULL PAPER
excess of an external thiol (50 equiv. MESNa) was added to well as other unusual amino acids by thiol-ene reactions
facilitate the formation of the MES thioester to generate to generate a molecular diversity found in many naturally
SFTI-MES 11b in 54% yield after 24 h. In addition to the occurring peptides.[32] Racemization of MeCys is not a con-
thioester, two thiolactones 12a and 12b were also observed, cern in Nα-amide bond cleavage when it is used as a thioes-
which were derived by thiol/thioester exchange reactions of ter surrogate as MeCys is a leaving group in thioester for-
the internal and N-terminal cysteinyl thiols with the C-ter- mation. In Cα-amide bond cleavage, racemization of MeCys
minal thioester. These three products were used for the thia has not been studied but it is a concern in the preparation
zip cyclization at pH 7.5 for 2 h at room temperature (Fig- of N-alkylated peptides. One solution is to exploit the Me-
ure 6). To prevent disulfide formation, 20 mm tris(2-carb- Cys residue as a precursor of N-methyl-dehydroalanine,
oxyethyl)phosphine (TCEP) was added to the cyclization which diminishes the concern over racemization.
buffer. Once formed, a proximity-driven S–N acyl shift re-
The limitations of using MeCys as a thioester surrogate
action leads to the formation of an amide bond to generate include side-reactions such as the formation of diketopiper-
the desired amido macrocycle. Because SFTI-1 contains azine, β-elimination, and Cα-cleavage in strong acid. These
only one internal cysteine, the cyclization reaction was com- side-reactions can be avoided by eliminating the carbonyl
paratively slow and required 4 h for completion. Iodine oxi- group at the C-terminal side of the MeCys residue. Indeed,
dation under acidic conditions was used for disulfide for- we have taken advantage of this observation recently to de-
mation in cyclic SFTI and the excess iodine quenched by velop the thioethylbutylamido thioester surrogate, which re-
ascorbic acid. Thus, the synthesis of SFTI-1 by the reverse tains the advantage of MeCys and excludes the side-reac-
ligation process was achieved in 31% overall yield by using tions and direction of the acidic isomerization for preparing
methyl cysteinyl peptide precursors.
thioesters.[16]
Conclusions
Experimental Section
Synthesis of TIGGIR Series Peptides: Rink amide resin (0.34 mmol/
g, 0.1 mmol, 294 mg) or Wang resin (0.8 mmol/g, 0.1 mmol,
125 mg) was used to synthesize the peptides. The resin was first
swollen in DCM for 30 min and then deprotected with 20% piper-
idine in DMF for 5 min twice. After removing the Fmoc groups,
Fmoc-Gly-OH (MW = 297.3, 0.4 mmol 119 mg) was introduced
onto the resin by using N,NЈ-diisopropylcarbodiimide (DIC, MW
126.2, d = 0.806 g/mL, 0.4 mmol, 126 mL) and 1-hydroxy-7-aza-
benzotriazole (HOAT, MW 136.1, 0.4 mmol, 54 mg). Peptide
In this work we have demonstrated that MeCys mediates
a bi-directional peptide bond cleavage and its directionality
can be manipulated by controlling the acidity of the reac-
tion conditions. The directional cleavage is dependent on
the protonated state of the Cys thiol side-chain and the
scissile peptide bond.
Under aqueous conditions at pH 1–5, the MeCys (amide
form) in Xaa-MeCys-Yaa mediates Nα-cleavage of the Xaa-
MeCys bond through an N–S acyl shift to form an isomer elongation was carried out manually by using 4 equiv. Fmoc amino
acid, 4 equiv. (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP, MW 520.4), and 6 equiv. N,N-diiso-
propylethylamine (DIEA, MW 129.2, d 0.742 g/mL) in DMF for
30 min. Fmoc deprotection was performed by using 20% piperidine
in DMF (5 min ϫ2). The coupling and deprotection reactions were
monitored by means of the ninhydrin test. The coupling of amino
acids to the N-methyl amino acid-loaded resin was achieved by
using 4 equiv. Fmoc amino acid, 4 equiv. O-(7-azabenzotriazol-1-
yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate (HATU,
MW 380.2), and 6 equiv. DIEA in DMF for 45 min twice. The
(thioester form) and in tandem with thiol/thioester ex-
change reaction to form another thioester (Xaa-thioester)
or a thiolactone (Xaa-thiolactone). At pHϾ6, such a thio-
ester or thiolactone can undergo native chemical ligation to
form a new peptide bond with an N-terminal Cys residue,
as demonstrated by our examples using TIGGIR as a
model peptide. This strategy is particularly useful for the
synthesis of a cysteine-containing cyclic peptide such as
SFTI in which both the forward (N–S acyl shift) and re-
verse (S–N acyl shift) reactions are exploited for the synthe- coupling reaction was monitored by means of the acetaldehyde/
chloranil test. After peptide chain assembly, the protected peptide
was treated with TFA/triisopropylsilane (TIS)/H2O (9.5 mL/
250 μL/250 μL) for 2 h. Then the crude peptide was precipitated
with cold diethyl ether and the precipitate was dried in vacuo and
later purified by reversed-phase (RP)-HPLC using Grace prepara-
tive columns.
sis of a peptide thioester and subsequently a cyclic peptide
in a ligation reaction.
Under strongly acidic conditions at H0 = –5, MeCys
(amide form) also mediates Cα-cleavage of the MeCys-Yaa
bond via an oxazolone intermediate to form a C-terminal
thiocresol thioester, which is readily converted into a β-
thiolactone. One interesting aspect of this reaction is that
the thioethylamido moiety, as described previously, partici-
pates in a displacement reaction in both directions, resulting
in either a thioester or a β-thiolactone. β-Thiolactones have
been little studied, but are potentially useful for the synthe-
sis of thiocarboxylic acid in peptide synthesis. Furthermore,
a MeCys-containing peptide is a useful synthetic precursor
for the preparation of MeAla through a desulfurization re-
Synthesis of TIGGIRC-TEBA: Cl-Trt(2-Cl) resin (167 mg,
0.2 mmol) was swollen with CH2Cl2 for 30 min and washed with
CH2Cl2 (5 mL ϫ2). (2-Butylamino)ethanethiol
1 (15.0 μL,
0.1 mmol) in CH2Cl2 (10 mL) were added and the suspension was
shaken for 1 h at room temp. Then DIEA (106 μL, 0.6 mmol) in
MeOH (5 mL) was added to the suspension and shaken for 10 min.
The resin was filtered, washed with CH2Cl2 (1 min ϫ3) and DMF
(1 min ϫ3). Fmoc-Cys(Trt)-OH (MW 585.3, 0.4 mmol, 234 mg)
was introduced onto the resin by using DIC (0.4 mmol, 126 μL)
action and N-methyl-dehydroalanine by β-elimination, as and 1-hydroxy-7-azabenzotriazole (HOAt, MW 136.1, 0.4 mmol,
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Amide Bond Cleavage of N-Methylcysteinyl Peptide
54 mg). The resin was filtered and washed with DMF. The coupling
reaction was monitored by means of the acetaldehyde/chloranil
test to detect a secondary amino group. Peptide elongation was
carried out manually by using Fmoc amino acid/PyBOP/DIEA
(4:4:6 equiv.) in DMF for 30 min. Fmoc deprotection was per-
formed by using 20% piperidine in DMF (5 min ϫ2).
tored by RP-HPLC and MALDI-TOF MS. The ligation product
was analyzed by 4800 MALDI-TOF MS followed by MS/MS.
TFMSA Treatment of the TIGGIR Series Peptides: Peptide
(0.2 μmol, 150–180 μg) was dissolved in TFA (170 μL) and then 1%
trifluoromethanesulfonic acid (TFMSA, 20 μL) and TC or MMA
(10 μL) were added. The reaction mixture was shaken at room
temp. for 2 h. The peptide was then precipitated by cold dimethyl
ether and dissolved in Milli-Q H2O. The peptide solution was sub-
jected to analytical HPLC immediately.
Synthesis of SFTI-MeCys-Gly-NH2: Rink amide resin (0.34 mmol/
g, 0.1 mmol, 294 mg) was used to synthesize the C-terminal amide
peptides. The resin was first swollen in DCM for 30 min and then
deprotected with 20% piperidine in DMF (5 min ϫ2). After re-
moving the Fmoc groups, Fmoc-Gly-OH (MW 297.3, 0.4 mmol,
119 mg) was introduced onto the resin by using DIC (0.4 mmol,
63 mL) and HOAt (0.4 mmol, 54 mg). The MeCys residue was cou-
pled by using Fmoc amino acid/PyBOP/DIEA (4:4:6 equiv.) in
DMF for 30 min. Fmoc deprotection was performed by using 20%
piperidine in DMF (5 min ϫ2). The coupling and deprotection re-
actions were monitored by using the ninhydrin test. The amino acid
after N-methyl amino acid was coupled by using Fmoc amino acid/
HATU/DIEA (4:4:6 equiv.) in DMF (45 min ϫ2). The coupling
reaction was monitored by means of the acetaldehyde/chloranil
test. Then the resin was subjected to the CEM Liberty 1 automated
microwave peptide synthesizer to perform the peptide elongation
reaction using PyBOP/DIEA for the coupling and 20% morpholine
in DMF for the deprotection. Following the peptide chain as-
sembly, 448 mg of dry resin was obtained. Then the side-chain-
protected peptide was treated with TFA/TIS/H2O (9.5 mL/250 μL/
250 μL) for 2 h and the crude peptide was precipitated with cold
diethyl ether. Later, the precipitate was dried in vacuo and purified
by RP-HPLC using a preparative column. SFTI-MeCys-Gly-NH2
was afforded in 17% isolated yield.
Nucleophile Treatment of β-Thiolactone: A 1 m NH2OH/HCl (MW
69) or NH2NH2/H2O solution (MW 50) was added to the fraction
containing β-thiolactone from HPLC to give a final concentration
of 100 mm. Then the reaction mixture was adjusted by the addition
of 1 m NaOH to pH 7 and the reaction allowed to proceed at room
temp. for 5 min. The products were analyzed by MALDI-TOF MS
followed by MALDI-TOF MS/MS.
S-Alkylation of TR6-MeCys-TC: Peptide TIGGIR-MeCys-S-TC
was dissolved in 200 mm citric buffer (pH 3.0) containing 20 mm
N-ethylmaleimide (NEM) to give a final peptide concentration of
1 mm. S-alkylation was performed at room temperature for 1 h.
Excess NEM was removed by RP-HPLC purification to give the
TIGGIR-MeCys (NEM)-S-TC thioester.
Preparation of TR6-Cys-TC and TR6-Cys-MMA: TIGGIR-Cys-
TEBA (MW 834) was dissolved in acetic acid to give a final con-
centration of 5 mm along with 50 equiv. of TC and incubated at
40 °C for 24 h. Acetic acid was then evaporated by Thermo Scien-
tific Speedvac SPD 1010. The remaining peptide was precipitated
by cold dimethyl ether and purified by RP-HPLC to afford TR6-
Cys-TC.
N–S Acyl Shift and S–S Thiol Exchange Under Aqueous Conditions:
Buffer solutions with pH values in the range of 1–7 were prepared.
Solutions of pH 1 and 2 were prepared by using diluted sulfuric
acid at concentrations of 0.1 and 5 mm, respectively. Solutions of
pH 3, 4, and 5 were prepared using 0.1 m sodium phosphate buffer.
Solutions of pH 6 and 7 were prepared by using 0.2 m sodium phos-
phate buffer containing 20 mm TCEP to prevent unwanted disul-
fide formation. TIGGIR-MeCys-Gly-NH2 (MW 789) was dis-
solved in the buffer to give a final concentration of 5 mm with
50 equiv. of external thiol MESNa and incubated at 40 °C. Ali-
quots were withdrawn after different periods of time and analyzed
by RP-HPLC at 220 nm.
TIGGIR-Cys-TEBA was dissolved in the buffer to give a final con-
centration of 5 mm along with 50 equiv. of external thiol MMA and
incubated at 40 °C. The peptide was then purified by RP-HPLC to
afford TR6-Cys-MMA.
Determination of the Acidity of a 0.1% TFMSA/TFA Solution: The
acidity of TFMSA/TFA anhydrous solutions was determined from
the protonation ratio of dimethyl sulfide (DMS) dissolved in the
acid solutions. The equilibrium of protonated and unprotonated
DMS was measured by using 1H NMR spectroscopy.[24] DMS was
not ionized in TFA (H0 = –3.03) but started to be protonated at
higher acidities.
Thioester-Capture Peptide Ligation: Peptide thioester TIGGIR-
MES (1 mm) was captured by the N-terminal Cys of synthetic pept-
ide CALVIN (3 mm) to give ligation product TIGGIRCALVIN in
0.2 m sodium phosphate buffer solution (pH 7.5) containing 6 m
guanidine·HCl and 20 mm TCEP. The reaction was performed with
50 equiv. external thiol methyl mercaptoacetate (MMA) at room
temperature for 3–6 h. Ligation products were purified by RP-
HPLC.
A series of TFMSA/TFA solutions with 0–10% (v/v) TFMSA, 5%
DMS (substrate), and 5% TMAS (tetramethylammonium sulfate;
internal standard) were prepared on ice and analyzed by 1H NMR
spectroscopy at 400 MHz. The chemical shift of TMAS [ν(IS)] was
used to auto-zero the NMR spectra. The chemical shifts of nonpro-
tonated [Δν(B)] and fully protonated DMS [Δν(B+)] were acquired
in pure TFA and 10% TFMSA/TFA solutions, respectively. The
ionization ratio (I) was calculated from the change in the chemical
shift by using Equation (1).
One-pot Peptide Ligation: Peptides and TIGGIR-MeCys-Gly-NH2
were dissolved in 0.2 m sodium phosphate buffer with 6 m guanid-
ine hydrochloride and 20 mm TCEP (pH 5) to prepare a 1 mm solu-
tion. Then 1 equiv. CALVIN was added together with 50 equiv.
MMA and the reaction mixture was incubated at 40 °C to give the
ligation product TIGGIRCALVIN.
Δν(DMS) – Δν(B)
I =
(1)
Δν(B+) – Δν(B)
By calculating the ratio I in each TFMSA solution, the TFMSA
concentration (vol.-%) was plotted against DMS protonation ratio
(%), which showed a linear correlation between protonation ratio
and acidity.
Ligation Reaction of TR6-MeCys-TC and CALVIN: Peptide thioes-
ter TR6-MeCys-TC (1 mm) was captured by the N-terminal Cys-
SH of the synthetic peptide CALVIN (3 mm) to give ligation prod-
uct TIGGIR-MeCys-CALVIN in 0.1 m sodium phosphate buffer
solution (pH 7.5) containing 6 m guanidine·HCl and 10 mm TCEP.
The reaction was performed at room temperature for 2 h and moni-
The acidity function H0 was calculated by using Equations (2) and
(3) in which m = 1.26 and pKBH+ = –6.95.[33]
Eur. J. Org. Chem. 2014, 4370–4380
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logI = –mH0 + pKBH+
(2)
(3)
1
H0 = –
(6.95 + logI)
1.26
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Supporting Information (see footnote on the first page of this arti-
cle): Characterization data for all model peptides, HPLC and mass
spectrometry traces for the synthetic intermediates.
Acknowledgments
This research was supported in part by the Biomedical Research
Council (BMRC) (grant number 09/1/22/19/612) of A*STAR and
the National Research Foundation of Singapore (grant number
CRP8-2011-05).
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Received: March 14, 2014
Published Online: June 6, 2014
4380
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