An fmoc compatible, O to S shift-mediated procedure for the preparation of C-terminal thioester peptides

Article abstract of DOI:10.1021/jo4015112

We report a practical 2-hydroxy-3-mercapto-propionic acid (Hmp)/2-methylpiperidine (2-MP) based Fmoc chemistry procedure to prepare the C-terminal Hmp peptides, which serve as the precursors of C-terminal thioester peptides. The subsequent O to S acyl shift and thiol-exchange mediated thioester conversion of the crude precursor peptides can be accomplished smoothly under mild conditions to provide the desired thioester peptides with good yield and high quality. This is a highly adaptable approach, and we envision its broad application in the preparation of C-terminal thioester peptides.

Full text of DOI:10.1021/jo4015112

Article
pubs.acs.org/joc
An Fmoc Compatible, O to S Shift-Mediated Procedure for the
Preparation of C‑Terminal Thioester Peptides
Fa Liu* and John P. Mayer
Lilly Research Laboratories, Indianapolis, Indiana 46285, United States
S
* Supporting Information
ABSTRACT: We report a practical 2-hydroxy-3-mercapto-propionic acid (Hmp)/2-methylpiperidine (2-MP) based Fmoc
chemistry procedure to prepare the C-terminal Hmp peptides, which serve as the precursors of C-terminal thioester peptides.
The subsequent O to S acyl shift and thiol-exchange mediated thioester conversion of the crude precursor peptides can be
accomplished smoothly under mild conditions to provide the desired thioester peptides with good yield and high quality. This is
a highly adaptable approach, and we envision its broad application in the preparation of C-terminal thioester peptides.
tion.59 However, despite the advantages of the Hmp process,
INTRODUCTION
■
the current methods49−52,60−62 are suboptimal because of their
Building upon the well-established techniques of solid-phase
low yields and synthetic complexity, which have limited their
peptide synthesis,1,2 the seminal development of native
practical application. To facilitate broader utilization of the
chemical ligation (NCL) by Kent and co-workers has enabled
Hmp procedure, we report a practical, Fmoc compatible
the efficient assembly of longer peptides (>50 residues) and
method to prepare C-terminal Hmp peptides. In addition, we
even of small to medium size proteins.3 The C-terminal
present the results of a systematic examination of the influence
thioester, a key component of NCL, has been traditionally
of the residue P(−1) (the residue coupled to Hmp) on the
prepared through Boc/Bzl chemistry. However, the required
thioester yield. Finally, we also disclose a validated, convenient
use of hydrogen fluoride as well as the incompatibility with
one-pot protocol to generate the final peptide thioesters for use
certain synthetic modifications has limited the use of the Boc/
Bzl approach and has prompted the search for Fmoc/t-Bu
in ligation.
based methods. Preparation of peptide C-terminal thioesters by
conventional Fmoc/t-Bu chemistry is compromised by the
RESULTS AND DISCUSSION
■
instability of the thioester during the Fmoc deprotection step
(20% piperidine in dimethylformamide, DMF). To overcome
this challenge, numerous alternative approaches have been
explored over the past decade.4−54 Most recently, the unique
intramolecular N to S33−47 and O to S48−54 acyl shift mediated
methods have gained favor, and associated new procedures
continue to be reported. In each case, a precursor peptide is
synthesized and then is converted to the desired thioester either
in situ or prior to the ligation. The Hmp (2-hydroxy-3-
mercapto-propionic acid) mediated intramolecular O to S acyl
shift, in particular, attracted our attention by virtue of its fast
conversion rate, clean product distribution, and mild reaction
conditions.49,50 These characteristics are essential for producing
homogeneous, high-quality peptides for pharmaceutical appli-
cations. It is known that certain existing post solid-phase
treatments have been shown to cause C-terminal epimeriza-
tion8,29,55 or side chain modifications.29,56−58 In addition, the
relatively harsh reaction conditions (too acidic or basic,
extended reaction time, or high temperatures (>37 °C))
utilized in some procedures contribute to peptide degrada-
Synthetic Methods of Hmp Peptides. Botti et al.’s
elegant approach of directly oxidizing the resin-bound Cys63 to
the corresponding Hmp is compromised by the inefficiency of
on-resin diazotization/hydrolysis (Supporting Information
Scheme S1).49 While George et al. have overcome this
limitation by preforming the 2-(t-butyl-dimethyl-silanyloxy)-3-
t-butyldisulfanyl-propionic acid building block, the utility of this
multistep procedure is limited by its complexity (Supporting
Information Scheme S2).52 To further optimize the synthetic
procedure for conciseness and practicality, we have developed
an alternative in the form of a trityl protected Hmp linker 364,65
which can be readily obtained via a single-step, regiospecific
epoxide ring-opening of the commercially available potassium
oxirane-2-carboxylate 1 with sodium triphenylmethanethiolate
in DMF (Scheme 1). After a simple workup involving reverse
extraction, acidification, and re-extraction, 3 was obtained in
51% yield and was ready for use without additional purification.
Received: July 18, 2013
© XXXX American Chemical Society
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Scheme 1. Hmp Based Procedure for the Preparation of C-Terminal Thioester Peptide
3 can be efficiently coupled without protection of the α-
hydroxyl group to commercial Rink amide resin under standard
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-
fluorophosphate (HBTU)/hydroxybenzotriazole (HOBT)/Dii-
sopropylethylamine (DIEA)66 conditions to provide resin 4. A
two-step solid-phase procedure was initially attempted to
prepare 4 by coupling 1 to the resin followed by opening of the
resin-bound epoxide with sodium triphenylmethanethiolate.
Although this procedure yielded the desired product when
preparing the random model peptide 6-Gly (YQHVFLGHmp-
NH2, Table 1), a side product with a 16 Da lower molecular
weight was consistently identified during several test syntheses
(Supporting Information Figure S1). This side product was
presumably the result of a competing epoxide ring-opening by
the resin-bound amine group during the loading step.
The subsequent coupling the of Fmoc-protected Gly, Ala,
and Tyr(t-Bu) residues to the α-hydroxyl group of resin-bound
Hmp proceeded efficiently by employing a standard HBTU/
HOBT/DIEA/DMAP(cat.) protocol. Assembly of the com-
plete sequence by means of automated peptide synthesis
followed by cleavage using standard trifluoroacetic acid (TFA)/
triisopropylsilane (TIS)/H2O treatment successfully afforded
model peptides 6-Xaa (Xaa: Gly, Ala, and Tyr). These
conditions, however, gave poor results in the case of the C-
terminal β-branched residue Val. In the course of investigating
an alternative method for coupling Val, use of the Mitsunobo
reaction67−70 was shown to give dramatically improved yields
(Supporting Information Figure S2). The efficiency of the
Mitsunobu coupling method was then assessed for all naturally
occurring amino acids with the exception of Cys. The efficiency
was found to be acceptable for the majority of Fmoc-protected
residues, however, Arg(Pbf), Glu(t-Bu), Pro, Asp(t-Bu),
Asn(Trt), and Trp(Boc) gave less than satisfactory results
presumably because of their bulky side-chain protecting groups
as well as the tendency of Pro to undergo diketopiperazine
formation. We suspect that the sterically hindered side-chains
may have restricted access of the α-carboxy group to the
triphenylphosphine activated α-hydroxyl of the resin-bound
Hmp. A variety of conditions were examined for coupling
Arg(Pbf), Glu(t-Bu), Asp(t-Bu), Asn(Trt), and Trp(Boc) to
Hmp, including the use of symmetric anhydrides (at room
temperature (rt) or 60 °C), the bis-(trichloromethyl) carbonate
(BTC) mediated in situ generation of acyl chloride,71
trichloroacetimidate activation,72 HBTU/HOBT/DIEA/
DMAP(cat.), and the Mitsunobu reaction using PMe3 in
place of the sterically hindered PPh3.69 Surprisingly, only the
HBTU/HOBT/DIEA/DMAP(cat.) method worked effectively,
although the yields for Asn(Trt), Asp(t-Bu), and Arg(Pbf)
remained modest. Most of the model peptides 6-Xaa for the
investigation of O to S acyl shift process were purified by RP-
HPLC reverse phase-high performance liquid chromatography;
the crude 6-Asn, 6-Asp, and 6-Arg materials were lyophilized
and were used directly because of the low recovery (Table 1).
Some of the Hmp peptides 6-Xaa eluted as two close peaks on
the RP-HPLC corresponding to the two diastereoisomers
Table 1. Yield and Characterization of the Model Hmp
Peptides 6-Xaa (YQHVFLXaaHmp-NH2)
ESI-MS (M + H)+
amount
(mg)
obtained
from 0.10
mmol
residue
purity
(P−1)
by RP- coupling
a
b
peptide
synthesis
yield expected observed HPLC method
6-Ala
6-Asp
6-Glu
6-Phe
6-Gly
6-His
6-Ile
51
<5
30
30
25
30
35
30
30
30
<5
30
<5
30
30
30
30
70
52%
<5%
29%
28%
26%
29%
34%
29%
29%
29%
<5%
29%
<5%
30%
30%
27%
28%
69%
980.5
1024.4
1038.5
1056.5
966.4
980.3
1024.3
1038.2
1056.3
966.2
93%
76%
95%
90%
95%
90%
96%
96%
92%
85%
70%
92%
55%
94%
90%
95%
90%
96%
2
1
1
2
2
2
2
2
2
2
1
2
1
2
2
1
2
2
c
1046.5
1022.5
1037.5
1022.5
1040.5
1023.4
1036.5
1065.5
996.5
1046.3
1022.3
1037.3
1022.3
1040.3
1023.2
1037.3
1065.3
996.2
6-Lys
6-Leu
6-Met
6-Asn
6-Gln
6-Arg
6-Ser
6-Thr
6-Trp
6-Tyr
6-Val
c
c
1010.5
1095.5
1072.5
1008.5
1010.3
1095.3
1072.3
1008.3
a
All of 6-Xaa were repared by using piperidine as the Fmoc removal
b
agent. Method 1: HBTU/HOBT/DIEA/DMAP(cat.); method 2:
Mitsunobu reaction. Crude peptides.
c
B
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Table 2. Yield and Characterization of the Model Hmp Peptides 7-8
c
peptide number
sequence
yield
expected MW
observed MW
purity by RP-HPLC
a
7-Ala
ADEFGHIKLMNPQRSTVYAHmp-NH2
ADEFGHIKLMNPQRSTVYGHmp-NH2
ADEFGHIKLMNPQRSTVYGHmp-NH2
ADEFGHIKLMNPQRSTVYQHmp-NH2
ADEFGHIKLMNPQRSTVYVHmp-NH2
ADEFGHIKLMNPQRSTVYYHmp-NH2
17%
9%
2280.6
2266.6
2266.6
2337.6
2308.6
2372.7
2652.0
2279.8
2266.4
2266.4
2337.4
2308.4
2372.2
98%
98%
98%
98%
98%
98%
95%
a
7-Gly
b
7-Gly
39%
28%
34%
27%
25%
a
7-Gln
a
7-Val
a
7-Tyr
a
8
ADEFGHIKLMN(Ac3AcNH-β-Glc)PQRSTVYLHmp-NH2
2651.6
a
b
c
Fmoc was removed by piperidine (11 min). Fmoc was removed by double 2-MP treatments (11 min × 2). After manual deconvolution of the
ESI-MS signals.
resulting from the racemic 3 used in the peptide synthesis
(Supporting Information Figure S6).
Table 3. Sequences of the Model Hmp Peptides 6-Xaa and
Their Resulting C-Terminal Thioester Peptides 9-Xaa and
10
To test the practical utility of this method, we prepared an
additional set of longer, random model peptides 7-Xaa
(ADEFGHIKLMNPQRSTVYXaaHmp-NH2, Xaa: Ala, Gln,
Gly, Tyr, or Val) as well as the N-linked glycopeptide 8
(ADEFGHIKLMN(Ac3AcNH-β-Glc)PQRSTVYLHmp-NH2).
Resin cleavage in all cases provided crude products with
comparable analytical purities to that obtained from conven-
tional synthesis of similar length of peptides, however, in
somewhat lower yield (Table 2 and Supporting Information
Figure S7). In one instance, the yield was 9% for peptide 7-Gly
with a Gly at residue P(−1). In another example, the cleavage
of a 40-mer C-terminal Hmp peptide with Gly at P(−1) yielded
no peptide material. This was presumably a consequence of the
instability of the oxo-ester bond between residue P(−1) and
Hmp during Fmoc deprotection involving treatment with 20%
piperidine in DMF for 11 min for each cycle (total exposure:
7.5 h for a 40-mer peptide). To confirm this hypothesis, the
resin-bound peptide 6-Gly was treated with 20% piperidine in
DMF, and the peptide release was monitored through analysis
of the DMF supernatant using liquid chromatography-mass
spectrometry (LC-MS) (Supporting Information Figure S3).
We found that these conditions effectively resulted in
aminolysis and premature release of peptide YQHVFLG-
N(CH2)5 from the resin. The aminolysis rate appeared to be
residue P(−1) dependent on the basis of the yields of peptides
7-Xaa with Gly being the most susceptible. To overcome this
limitation, we selected the more sterically hindered 2-MP (2-
methyl piperidine)73,74 in the deprotection step as an
alternative to piperidine. When testing 2-MP in place of
piperidine in the above protocol, we detected no aminolysis
even after a 24 h treatment period (an exposure time equivalent
to a 65-residue peptide synthesis with double 2-MP treatments
per cycle) (Supporting Information Figure S3). The resynthesis
of 7-Gly employing a double 2-MP treatment (11 min × 2) for
Fmoc deprotection afforded crude product with identical purity
as 7-Gly prepared using piperidine but in a dramatically
improved yield of 39% after RP-HPLC purification (Table 2,
Figure 4, and Supporting Information Figure S7).
peptide
number
sequence
6-Xaa YQHVFLXaaHmp-NH2 (Xaa: Ala, Arg, Asn, Asp, Gln, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val)
9-Xaa YQHVFLXaa-S(CH2)2CO2H (Xaa: Ala, Arg, Asn, Asp, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val)
10
YQHVFLG-S(1,4-C6H4)CH2CO2H
The conversions were performed in the commonly used 0.20
M sodium phosphate buffer containing 6 M Gn (guanidine).
Model peptide 6-Gly was used to assess the influence of pH on
thioester yield with 2% aliphatic MPA (3-mercaptopropionic
acid) or 50 mM of the aromatic MPAA (4-mercaptophenyl-
acetic acid) as the free thiol source (Figure 1, Supporting
Information Tables S1 and S2). No conversion for either thiol
was observed at or below pH 3 at the 6 h time point.
Measurable conversion did occur at pH 5, although the rate was
relatively low. At pH 6, however, the conversion was completed
within 30 min for MPAA and within 90 min for MPA, while at
pH 7 or higher, both reactions finished within 5 min. MPA
ester 9-Gly (YQHVFLG-S(CH2)2CO2H) was reasonably stable
at pH 8 or below; however, the hydrolysis of the corresponding
MPAA ester 10 (YQHVFLG-S(1,4-C6H4)CH2CO2H) became
significant at pH 6.5 and higher (Figure 1). Because of the
lower stability of the MPAA esters, we focused on the MPA
esters when examining the influence of residue P(−1). In
addition to C-terminal amide peptide 6-Gly, the C-terminal
acid peptide 11 (YQHVFLGHmp) was also prepared and
tested. However, the C-terminal acid 11 was significantly more
prone to hydrolysis (∼85%) when compared to the amide 6-
Gly (∼20%) in the O to S acyl shift step.
In the following experiment, peptides 6-Ala, 6-Tyr, and 6-
Val were tested at pH 6.5, 7.0, 7.5, and 8.0 in 2% MPA, 6 M
Gn, and 0.2 M sodium phosphate buffer (Supporting
Information Tables S3−S5 and Figure S4). At pH 7.0 and
above, the thioester formation was judged complete within 30
min for 6-Ala and 6-Tyr and within 60 min for 6-Val. The
resulting MPA thioesters 9-Xaa were stable under these
conditions with yields of around 80% for 9-Ala and 9-Tyr
but disappointingly of less than 30% for 9-Val. The modest
yield of 9-Val was presumably due to the steric influence of the
β-branched side chain which slowed the O to S acyl shift
sufficiently to allow the competing hydrolysis of the oxo-ester
to prevail to a significant degree. On the basis of this
hypothesis, we reasoned that one of the Hmp peptide
diastereoisomers may undergo acyl shift more efficiently than
the other. The racemic Hmp used in the synthesis yielded two
The Impact of pH and Residue P(−1) on the Yield of
Thioester Conversion. Having optimized the synthesis of the
Hmp peptides, we next investigated the efficiency of thioester
formation through O to S acyl shift and the subsequent thiol-
exchange reactions using model peptides 6-Xaa (Table 3). We
initially focused on the pH dependence of this transition and
later assessed the impact of the residue P(−1) on the thioester
yield at the optimal pH. Finally, we screened several solvent
systems to minimize the hydrolysis and to maximize the
thioester formation.
C
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Figure 1. The impact of pH on O to S acyl shift yield (6-Gly in 2% MPA or 50 mM MPAA, 6 M Gn, and 0.2 M sodium phosphate buffer).
Figure 2. The impact of stereochemistry of Hmp on thioester conversion yield of 6-Val to 9-Val (the spectra were collected at 220 nM).
Figure 3. The impact of residue P(−1) on the O to S acyl shift yield (6-Xaa in 2% MPA, 6 M Gn, and 0.2 M sodium phosphate buffer, pH 7.0).
diastereoisomers of peptide 6-Val, which resolved cleanly using
RP-HPLC, permitting purification of each to homogeneity
(Supporting Information Figure S2). However, the acyl shift
yields for the two 6-Val diastereoisomers were not dramatically
different: 26% versus 30% (Figure 2).
All remaining peptides 6-Xaa were tested at the fixed optimal
pH of 7.0 (Figure 3 and Supporting Information Table S6). To
summarize, the 2% MPA in 6 M Gn and 0.2 sodium phosphate
buffer (pH 7.0) condition afforded 70−80% yield of thioester
product for most of the P(−1) residues of model peptide 6-Xaa
while only 25−50% for the β-branced Val, Ile, and Thr.
To improve the O to S acyl shift efficiency, peptide 6-Val was
selected as a model to screen additional conditions. Because the
hydrolysis appeared to be the only side reaction, we initially
focused on anhydrous conditions. However, it was found that
when 6-Val was treated with thiols MPA, MESNa (sodium 2-
mercaptoethanesulfonate), β-mercaptoethanol, MPAA, or
thiophenol in anhydrous DMF, the result was either hydrolysis
D
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(with DIEA or imidazole as base) or no conversion (with
pyridine, 2,4,6-lutidine, or no base). We then turned to a
mixture of organic and aqueous solvent using various
concentrations of NMP (N-methylpyrrolidone) and 2% MPA
in 6 M Gn and 0.2 M sodium phosphate buffer (pH 7.0).
Surprisingly, we found that 100% aqueous conditions gave the
best yield (28%) and that no desired thioester was observed
when the aqueous component was lower than 50%. With these
results, we re-examined the original aqueous condition, and we
tested the impact of each buffer component (Supporting
Information Figure S5). The results suggested that 10% MPA
in 6 M Gn and 0.2 M sodium phosphate buffer (pH 7.0) was an
optimal condition, which gave the best yield of 56% for 6-Val
and which also provided excellent solubility for the peptides.
The thioester formation efficiency was then re-examined for
all the model peptides 6-Xaa under this optimized condition.
The yield for 9-Val and 9-Thr had been significantly improved
to 56% and 77%, respectively, while the yields of the additional
peptides had been modestly increased to a range of 80−90%,
the only exception being 9-Ile which remained at 30% (Table
4).
Information Figure S8A and S8B). To validate this approach,
the crude 7-Gly (prepared by the 2-MP method with double
treatments) obtained directly from resin cleavage was treated
with 10% MPA in 6 M guanidine and 0.2 M sodium phosphate
(pH 7.0) to facilitate the O to S acyl shift and thiol-exchange
and was judged to be completely converted within 20 min by
LC-MS. Subsequent RP-HPLC purification provided the
desired peptide C-terminal MPA thioester 12 in 38% yield
after lyophilization (Figure 4, Table 5, and Supporting
Information Figure S9). A 34-mer thioester 13 with Phe at
the C-terminus (SVSEIQLMHNLGKHLNSMER-
VEWLRKKLQDVHNF-S(CH2)2CO2H, N-terminal 34 resi-
dues of human parathyroid hormone) was succefully prepared
by this method with 28% overall yield and high purity as
confirmed by LC-MS (Figure 4, Table 5, and Supporting
Information Figure S9). Interestingly, although 2-MP was
considered less effective for Fmoc removal than piperidine,73,74
we found that a single 2-MP treatment (11 min) actually gave
identical crude purity as the double treatments when preparing
12 (Figure 4, Table 5, and Supporting Information Figure S9).
The efficiency of single 2-MP treatment was further confirmed
by the successful preparation of a 35-mer peptide thioester 14
with Gly at the C-terminus (SVSEIQLMHNLGKHLNSMER-
VEWLRKKLQDVHNFG-S(CH2)2CO2H) with a 14% yield
and excellent purity as determined by LC-MS (Figure 4, Table
5, and Supporting Information Figure S9).
Table 4. Yield of Conversion of Model Peptides 6-Xaa to
Thioester 9-Xaa under the Optimal Conversion Condition:
10% MPA in 6 M Gn and 0.2 M Sodium Phosphate Buffer
(pH 7.0)
peptide
yield
peptide
yield
peptide
yield
CONCLUSION
■
9-Ala
9-Asp
9-Glu
9-Phe
9-Gly
9-His
85%
83%
83%
80%
90%
90%
9-Ile
30%
82%
85%
84%
80%
86%
9-Arg
9-Ser
9-Thr
9-Trp
9-Tyr
9-Val
80%
85%
77%
80%
83%
56%
In conclusion, we have developed a practical, Hmp/2-MP based
Fmoc chemistry procedure enabling efficient preparation of C-
terminal Hmp peptides. Subsequent O to S acyl shift and thiol-
exchange-based conversion of the crude precursor to the
thioester can be quickly accomplished under very mild
conditions to afford the desired thioester after a single RP-
HPLC purification in good yield. We believe this optimized
Hmp/2-MP procedure is an effective and readily adaptable
method for the preparation of homogeneous peptide C-
terminal thioesters, and we envision its utilization in NCL and
other applications75−81requiring peptide thioesters.
9-Lys
9-Leu
9-Met
9-Asn
9-Gln
One-Pot Hmp/2-MP Based Method for C-Terminal
Thioester Peptide. Although the Hmp peptides can be used
directly for the native chemical ligation, the competing
hydrolysis of the Hmp peptides during the ligation is a
significant limitation of the existing methods, which hinders the
progress of the reaction and isolation of the final products (see
in situ ligation example in Supporting Information Figure
S8A).49−52 In addition, the two diastereoisomer Hmp peptides
formed from racemic Hmp 3 elute closely on RP-HPLC (rather
than eluting as a single component) which complicates their
purification. To address these constraints as well as to simplify
the overall process, we conducted the O to S acyl shift and the
subsequent thiol-exchange using the crude Hmp peptide
immediately following resin cleavage to provide the requisite
thioester peptides for the peptide ligation after a single RP-
HPLC purification (Scheme 2). This procedure also ensures
that the thioester conversion takes place at the optimal
condition to maximize the yield (see examples in Supporting
EXPERIMENTAL SECTION
■
General Information. All solvents [N,N′-dimethylforamide
(DMF), methanol (MeOH), dichloromethane (DCM), acetonitrile
(ACN), diethyl ether (Et2O), etc.] and reagents [hydroxybenzotriazole
(HOBT), N,N′-diisopropylcarbodiimide (DIC), O-(benzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU),
piperidine, 2-methylpiperidine (2-MP), trifluoroacetic acid (TFA), 3-
mercaptopropionic acid (MPA), 4-mercaptophenylacetic acid
(MPAA), thiophenol, sodium 2-mercaptoethanesulfonate (MESNa),
β-mercaptoethanol, potassium oxirane-2-carboxylate (vendor: Acesys
Pharmatech), guanidine (Gn), triisopropylsilane (TIS), diethyl
azodicarboxylate solution (40% in toluene) (DEAD), etc.] were
purchased and used directly. Water (H2O) was obtained from Milli-Q
water purification system (Millipore). Polystyrene Rink amide (RAM)
Scheme 2. One-Pot Hmp Based Procedure for the Preparation of C-Terminal Thioester Peptide
E
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Figure 4. Analytical HPLC traces of crude Hmp peptide 7-Gly and thioester peptides 12−14 (at 220 nm). Peaks marked with * are the desired
products; peaks marked with & are MPA related compounds (not peptides as determined by mass spectrometry).
Table 5. Yield and Characterization of the Model MPA Thioester Peptides 12−14
c
peptide number
sequence
yield
expected MW observed MW
purity by RP-HPLC
a
12
ADEFGHIKLMNPQRSTVYG-S(CH2)2CO2H
38%
36%
28%
14%
2251.5
2251.5
4205.8
4262.9
2251.0
2250.6
4205.4
4262.0
98%
98%
98%
b
12
ADEFGHIKLMNPQRSTVYG-S(CH2)2CO2H
a
13
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-S(CH2)2CO2H
b
14
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFG-S(CH2)2CO2H
98%
a
b
c
Fmoc was removed by double 2-MP treatments (11 min × 2). Fmoc was removed by single 2-MP treatment (11 min). After manual
deconvolution of the ESI-MS signals.
resin and Chlorotrityl Polystyrene resin were purchased from Rapp
Polymere GMBH. Fmoc chemistry based amino acid cartridges were
purchased from several vendors and were used directly. LC-MS
system: 1100 series liquid chromatography mass spectrometer, model
G1956A/B. General analytical RP-HPLC condition: All the samples
were analyzed by using a Waters SymmetryShield RP18 Column (cat.
no. 186000179, 3.5 μm, 4.6 × 100 mm.): a linear gradient from 6%
aqueous ACN (0.1% trifluoroacetic acid) to 60% aqueous ACN (0.1%
trifluoroacetic acid) over 14 min at a flow rate of 1.0 mL/minute.
2-Hydroxy-3-(triphenylmethyl)thio-propanoic Acid 3. Tri-
phenylmethanethiol (11.0 g, 40 mmol) was dissolved in anhydrous
DMF (150 mL) under the protection of dry N2 gas, and this solution
was cooled to 0 °C by an ice−water bath. NaH (60% in mineral oil, 1.6
g, 40 mmol) was added carefully through several portions, and the
resulting solution was stirred at 0 °C for an additional 15 min.
Potassium oxirane-2-carboxylate solid (2.60 g, 20 mmol) was added in
one portion, and the resulting reaction mixture was then gradually
warmed to rt and was stirred overnight before being poured into H2O
(500 mL) and being extracted with Et2O (100 mL × 3). The Et2O
layer was discarded, and the aqueous phase was acidified by 1 N HCl
to pH 3−4 (determined by pH strips) and was extracted by EtOAc
(100 mL × 3). The combined EtOAc was washed by brine and was
dried over anhydrous Na2SO4 for 2 h. EtOAc was then removed by
rotary evaporator to provide product 3 as a pale yellow solid (3.73 g)
in 51% yield, which was ready for use without further purification. ESI-
MS (+VE) m/z: 387.2 (M + Na)+. HR-ESI MS calcd for
C22H20NaO3S (M + Na)+, 387.1025; found, 387.1029. 1H NMR
(400 MHz, DMSO-d6) 7.35−7.15 (15H, m), 5.55 (1 H, bs), 3.85 (1
H, m), 2.32 (2 H, m). 13C NMR (100 MHz, DMSO-d6) 174.1, 144.9,
144.4, 130.0, 129.8, 129.6, 129.3, 128.6, 128.5, 128.3, 128.0, 127.6,
127.4, 127.2, 69.4, 66.2, 36.4.
The resulting resin 4 was washed by DMF (6 mL × 6) and Et2O (3
mL × 3).
General Procedure of Coupling Residue P(−1) by Mitsuno-
bu Reaction. The above resin 4 (160 mg, 0.10 mmol, prepared
through Hmp 3) was washed by anhydrous THF (3 mL × 3) to
remove any moisture and was treated with a mixture of Fmoc-Xaa−
OH (1.0 mmol), DEAD (40 wt % in toluene) (0.46 mL, 1.0 mmol),
and triphenylphosphine (262 mg, 1.0 mmol) in anhydrous THF (5
mL) at rt for 4 h. The resulting resin was washed by DMF (6 mL × 3).
General Procedure of Coupling Residue P(−1) by HBTU/
HOBT/DIEA/DMAP(cat.) Condition. The resin 4 (160 mg, 0.10
mmol, prepared through Hmp 3) was treated with a mixture of Fmoc-
Xaa−OH (1.0 mmol), HBTU (380 mg, 1.0 mmol), HOBt (135 mg,
1.0 mmol), DIEA (0.18 mL, 1.0 mmol), and a catalytical amount of
DMAP (1.2 mg, 0.01 mmol, 10% of resin substitution) in DMF (3
mL) for 2 h at rt. This coupling step was repeated once. The resulting
resin was washed by DMF (6 mL × 3).
Preparation of Resin 4 by Two-Step Solid-Phase Procedure.
Rapp Polymere Rink amide resin (300 mg, 0.20 mmol) was treated
with 20% piperidine/DMF (6 mL) at rt for 20 min to remove the
Fmoc protection. After DMF washes (6 mL × 6), the resulting resin
was mixed with potassium oxirane-2-carboxylate 1 (126 mg, 1.0
mmol), HBTU (380 mg, 1.0 mmol), HOBt (135 mg, 1.0 mmol), and
DIEA (0.18 mL, 1.0 mmol) in DMF (3 mL) at rt for 2 h and then was
washed by DMF (6 mL × 3). In a separate flask, a fresh sodium
triphenylmethanethiolate solution was prepared by adding triphenyl-
methanethiol (556 mg, 2.0 mmol) into a NaH suspension (60% in
mineral oil, 72 mg, 1.8 mmol) in anhydrous DMF (5.0 mL) at 0 °C
and by stirring for additional 15 min. The above resin was briefly
washed by anhydrous DMF (6 mL) to remove the moisture before
being mixed with the fresh sodium triphenylmethanethiolate solution
for 60 min at rt. The resulting resin 4 was then washed by DMF (6 mL
× 6) and Et2O (3 mL × 3). DIEA, DBU, and Et3N were also tried as
the base for the epoxide ring-opening by triphenylmethanethiol;
however, this conversion occurred efficiently only when NaH was
used.
Preparation of Resin 4 by Using 3. Rapp Polymere Rink amide
resin (300 mg, 0.20 mmol) was treated by 20% piperidine in DMF (6
mL) for 20 min to remove the resin-bound Fmoc group. After DMF
washes (6 mL × 6), the resin was treated with a mixture of HBTU
(380 mg, 1.0 mmol), HOBt (135 mg, 1.0 mmol), DIEA (0.18 mL, 1.0
mmol), and Hmp 3 (365 mg, 1.0 mmol) in DMF (3 mL) for 2 h at rt.
Peptide 6-Gly (YQHVFLGHmp-NH2). Starting with the resin 4
(160 mg, 0.10 mmol) prepared through Hmp 3 or the two-step solid-
F
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The Journal of Organic Chemistry
Article
phase procedure as described above, the next Gly was coupled by
mixing the resin with Fmoc-Gly-OH (300 mg, 1.0 mmol), HBTU
(380 mg, 1.0 mmol), HOBt (135 mg, 1.0 mmol), DIEA (0.18 mL, 1.0
mmol), and a catalytical amount of DMAP (1.2 mg, 0.01 mmol, 10%
of resin substitution) in DMF (3 mL) for 2 h at rt. This coupling step
was repeated once. The rest of the residues were assembled by using
peptide synthesizer ABI 433 with the standard Fmoc_HOBT_DCC
method (see the protocol details as described below). The resin
cleavage was conducted by treating the 0.10 mmol resin with 10 mL
TFA solution containing 2.5% TIS, 2.5% H2O at rt for 2 h with gentle
shaking. The resin was filtered off, and the filtrate was treated with 80
mL cold Et2O. The crude peptide was collected by centrifugation (see
Supporting Information Figure S1 for the analytical HPLC traces of
the crude products).
Peptide 6-Val (YQHVFLVHmp-NH2). Starting with the resin 4
(160 mg, 0.10 mmol, prepared through Hmp 3), the next Fmoc-Val-
OH was coupled by either HBTU/HOBT/DIEA/DMAP(cat.) or
Mitsunobu reaction as described above. The rest of the residues were
assembled by using peptide synthesizer ABI 433 with the standard
Fmoc_HOBT_DCC method (see the protocol details as described
below). The resin cleavage was conducted by treating the 0.10 mmol
resin with 10 mL TFA solution containing 2.5% TIS, 2.5% H2O at rt
for 2 h with gentle shaking. The resin was filtered off, and the filtrate
was treated with 80 mL cold Et2O. The crude peptide was collected by
centrifugation and was washed with cold Et2O (40 mL × 2) (see
Supporting Information Figure S2 for the analytical HPLC traces of
the crude products).
General Procedure for Synthesizing Model Peptides 6-Xaa,
7-Xaa, and 8. All the syntheses started with resin 4 (160 mg, 0.10
mmol, prepared through Hmp 3). The residue P(−1) was coupled by
using the Mitsunobu reaction. The rest of the residues were assembled
on the automatic peptide synthesizer ABI433 with standard
Fmoc_HOBT_DCC 0.10 mmol protocol or with the modified 2-
MP protocol (see Table 3 for details). The only difference between
these two protocols is that the deprotection step was repeated in 2-MP
protocol (11 min × 2 vs 11 min).
Resin cleavage was conducted by treating 0.10 mmol resin with 10
mL TFA solution containing 2.5% TIS, 2.5% H2O at rt for 2 h with
gentle shaking. The resin was filtered off, and the filtrate was treated
with cold Et2O (80 mL). The crude peptide was collected by
centrifugation, was washed with cold Et2O (40 mL × 2), and was
purified by preparative RP-HPLC using a Waters SymmetryPrep C18
Column (cat. no. WAT066245, 7 μm, 19 × 300 mm) with a linear
gradient from 5% aqueous ACN (0.05% TFA) to 50% aqueous ACN
(0.05% TFA) over 80 min at a flow rate of 15 mL/minute. The
peptides were eluted between 30 and 45 min depending on the
sequence. The final RP-HPLC fractions with desired product were
pooled together and were freeze-dried to afford the lyophilized
powder.
Oxo-Ester Bond (between Residue P(−1) and Hmp) Stability
Study with 20% Piperidine and 20% 2-MP in DMF. RAPP
Polymere resin bound-peptide 6-Gly (50 mg) was treated with 20%
piperidine or 20% 2-MP in DMF (1 mL). At each selected time point,
the supernatant (DMF phase) (50 μL) was removed and treated with
TFA (0.5 mL) for 20 min before diluted by ACN/H2O (1/1, 3 mL).
The resulting solution was subjected to LC-MS analysis (inject 50 μL);
the HPLC traces shown below are collected at wavelength 220 nM
(see Supporting Information Figure S3 for the analytical HPLC
traces).
Preparation of Peptide C-Terminal Acid 11 and the
Conversion to Thioester. Chlorotrityl polystyrene resin (0.20
mmol) was treated with Hmp 3 (0.40 mmol) and DIEA (1.0 mmol) in
DCM (3.0 mL) at rt for 2 h. After DMF washes (6 mL × 3), the resin
was treated with 5% piperidine in DMF (3 mL) to substitute all the
remaining trityl chloride. The resulting resin was treated with Fmoc-
Gly-OH (1.0 mmol), HBTU (1.0 mmol), HOBt (1.0 mmol), DIEA
(1.0 mmol), and a catalytical amount of DMAP (0.01 mmol, 10% of
resin substitution) in DMF (3 mL) for 2 h at rt. This step was
repeated once. After DMF washes (6 mL × 3), the rest of the residues
were assembled on the peptide synthesizer ABI433 using standard
Fmoc_HOBT_DCC method. The resulting peptide was treated with
2% MPA, 6 M Gn, and 0.2 M sodium phosphate buffer (pH 7.0) for
90 min and was analyzed by LC-MS, which indicated that 15% was
converted to the thioester and that 85% was hydrolyzed.
Assessment of Peptide Thioester Yield. Each peptide (1 mg/
mL) was dissoved in 6.0 M guanidine, 0.20 M sodium phosphate
buffer at the desired pH with 2% MPA or 50 mM MPAA. The sample
was then immediately monitored by LC-MS for up to 6 h. The yield of
peptide thioester was calculated by dividing the peptide thioester
HPLC peak area by the sum of the peptide thioester, the peptide acid
(hydrolyzed product), and any conversion intermediate. The further
optimization was conducted with 5 mg/mL peptide in each testing
condition with LC-MS monitoring the conversion progress (see
Supporting Information Tables S1−S6).
Impact of Stereochemistry of Hmp on O to S Acyl Shift Yield
by Using Two 6-Val Isomers. These two 6-Val isomers were
separated from the 6-Val prepared with racemic Hmp (see crude
analytical HPLC in Supporting Information Figure S2); each was
freeze-dried. The lyophilized powder was then treated with 2% MPA, 6
M Gn, and 0.2 M sodium phosphate at pH 7.0 (∼1.0 mg/mL peptide
concentration) at rt for 90 min to facilitate the O to S acyl shift and
thiol-exchange. The yield of resulting thioester was calculated by the
UV absorption areas at 220 nM with the baseline subtraction.
Examination of the Impact of Each Buffer Component on
the Thioester Formation Yield of 6-Val. Five milligrams/milliliter
of 6-Val was dissolved in the following buffers (all at pH 7.0): 2%
MPA, 6 M Gn, 0.2 M sodium phosphate; 10% MPA, 6 M Gn, 0.2 M
sodium phosphate; 20% MPA, 6 M Gn, 0.2 M sodium phosphate; 10%
MPA, 6 M Gn; 10% MPA, 0.2 M sodium phosphate; 10% MPA. The
samples were analyzed by LC-MS after 2 h at rt (see Supporting
Information Figure S5 for the analytical HPLC traces).
Table 6. Standard ABI433 Fmoc_HOBT_DCC Protocol and
the Modified 2-MP Protocol (Double Treatments)
12 (ADEFGHIKLMNPQRSTVYG-SCH2CH2CO2H) Prepared by
the One-Pot Hmp/2-MP Procedure with Double 2-MP Treat-
ment. Fmoc-Gly-OH was coupled to resin 4 (160 mg, 0.10 mmol,
prepared through Hmp 3) by the Mitsunobu reaction, and the rest of
the residues were assembled by ABI433 using the 2-MP protocol
(double treatments, 11 min × 2). The resulting resin (0.10 mmol) was
treated with TFA (9.5 mL), TIS (0.25 mL), and H2O (0.25 mL) for 2
h at rt. The resin was then filtered off, and the filtrate was treated with
cold Et2O (90 mL); the resulting crude peptide 7-Gly was collected by
centrifugation, was washed with cold Et2O (40 mL × 2), and was air-
dried (30 min). A freshly prepared 10% MPA in 6 M Gn, 0.2 M
sodium phosphate (pH 7.0) 5 mL was added to the dried crude 7-Gly.
This mixture was vortexed until fully dissolved and was kept at rt for
20 min before being subjected to LC-MS analysis which indicated that
the conversion to thioester had completed. This mixture was then
diluted by 0.1% TFA containing H2O (20 mL) and was purified by
preparative RP-HPLC column with the same conditions as described
above. The pooled fractions were freeze-dried to afford 86 mg 12 in
standard
Fmoc_HOBT_DCC
(0.10 mmol) protocol
2-MP (0.10 mmol) protocol
coupling
reagent
1 M DIC/DMF (1.0 mL) 1 M DIC/DMF (1.0 mL)
coupling
reagent
1 M HOBt/DMF
(1.0 mL)
1 M HOBt/DMF (1.0 mL)
1.0 mmol cartridge
Fmoc amino
acid
1.0 mmol cartridge
solvents
DMF and DCM
DMF and DCM
16 min
activation time 16 min
coupling time
37 min
37 min
deprotection
agent and
time
20% piperidine in DMF, 20% 2-MP in DMF, 11 min,
11 min
and 20% 2-MP in DMF,
11 min
G
dx.doi.org/10.1021/jo4015112 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
38% yield and 98% purity (by RP-HPLC), ESI-MS (+VE) m/z:
observed, 1126.5 (M + 2H)2+ (calculated: 1126.0).
(14) Ficht, S.; Payne, R. J.; Guy, R. T.; Wong, C.-H. Chem.Eur. J.
2008, 14, 3620.
12 (ADEFGHIKLMNPQRSTVYG-SCH2CH2CO2H) Prepared by
the One-Pot Hmp/2-MP Procedure with Single 2-MP Treat-
ment. 12 was prepared as previously described when preparing
thioester 12 (using double 2-MP treatment) except single 2-MP
treatment was used; the yield was 36%, and the purity was 98% by RP-
HPLC (40 mg of product was obtained from 0.05 mmol resin 4), ESI-
MS (+VE) m/z: observed, 1126.3 (M + 2H)2+ (calculated: 1126.0).
13 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-S-
(CH2)2CO2H) Prepared by the One-Pot Hmp/2-MP Procedure
with Double 2-MP Treatment. 13 was prepared as previously
described for the synthesis of thioester 12 (double 2-MP treatment) in
28% yield and 98% purity (by RP-HPLC) (118 mg of product was
obtained from 0.10 mmol resin 4), ESI-MS (+VE) m/z: observed,
1402.8 (M + 3H)3+ (calculated: 1402.9).
14 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFG-S-
(CH2)2CO2H) Prepared by the One-Pot Hmp/2-MP Procedure
with Single 2-MP Treatment. 14 was prepared as previously
described for the synthesis of thioester 12 (double 2-MP treatment)
except the single 2-MP treatment was used; the overall yield was 14%
with 98% purity (by RP-HPLC) (20 mg of product was obtained from
0.033 mmol resin 4), ESI-MS (+VE) m/z: observed, 1421.9 (M +
3H)3+ (calculated: 1422.0).
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ASSOCIATED CONTENT
* Supporting Information
■
S
1H and 13CNMR spectra of 3, the HPLC traces and mass
spectrometry data, the conversion kinetic study data of model
Hmp peptides 6-Xaa, and the example of native chemical
ligation by directly or stepwisely using C-terminal Hmp peptide
8. This material is available free of charge via the Internet at
http://pubs.acs.org
(34) Nakamura, K. i.; Sumida, M.; Kawakami, T.; Vorherr, T.;
Aimoto, S. Bull. Chem. Soc. Jpn. 2006, 79, 1773.
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Y.; Aimoto, S. Int. J. Pept. Res. Ther. 2007, 13, 191.
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Kawakami, T.; Aimoto, S. J. Pept. Sci. 2009, 15, 731.
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Nakahara, Y.; Nakahara, Y. Org. Lett. 2006, 8, 4465.
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A. Org. Lett. 2006, 8, 467.
AUTHOR INFORMATION
Corresponding Author
*E-mail: liufx@lilly.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Jason X. Tang (Eli Lilly) for his help with high
resolution mass spectrometry and Mr. Jeffry B. Franciskovich
(Eli Lilly) and Ms. Yen Dao (Eli Lilly) for their help with
NMR.
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