Thieme Chemistry Journals Awardees - Where Are They Now? Improved Fmoc Deprotection Methods for the Synthesis of Thioamide-Containing Peptides and Proteins

Article abstract of DOI:10.1055/s-0036-1589027

Site-selective incorporation of thioamides into peptides and proteins provides a useful tool for a wide range of applications. Current incorporation methods suffer from low yields as well as epimerization. Here, we describe how the use of 1,8-diazabicyclo

Full text of DOI:10.1055/s-0036-1589027

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© Georg Thieme Verlag Stuttgart · New York
2017, 28, A–F
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D. M. Szantai-Kis et al.
Letter
Synlett
Thieme Chemistry Journals Awardees – Where Are They Now?
Improved Fmoc Deprotection Methods for the Synthesis of
Thioamide-Containing Peptides and Proteins
D. Miklos Szantai-Kisa
‡
Christopher R. Waltersb
‡
Taylor M. Barrettb
Eileen M. Hoangb,c
E. James Petersson*a,b
a Department of Biochemistry and Molecular Biophysics,
Perelman School of Medicine, University of Pennsylvania,
3700 Hamilton Walk, Philadelphia, PA 19104, USA
ejpetersson@sas.upenn.edu
b Department of Chemistry, University of Pennsylvania, 231 S.
34th Street, Philadelphia, PA 19104, USA
c Swarthmore College, 500 College Ave, Swarthmore, PA
19081, USA
‡ These authors contributed equally to this work
Received: 12.03.2017
Accepted after revision: 10.04.2017
Published online: 10.04.2017
DOI: 10.1055/s-0036-1589027; Art ID: st2017-r0178-l
Abstract Site-selective incorporation of thioamides into peptides and
proteins provides a useful tool for a wide range of applications. Current
incorporation methods suffer from low yields as well as epimerization.
Here, we describe how the use of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) rather than piperidine in fluorenylmethyloxycarbonyl (Fmoc)
deprotection reduces epimerization and increases yields of thioamide-
containing peptides. Furthermore, we demonstrate that the use of DBU
avoids byproduct formation when synthesizing peptides containing
side-chain thioamides.
Key words peptides, sulfur, solid-phase synthesis, protecting groups,
proteins
E. James Petersson was educated at Dartmouth College, where he
worked in the laboratory of David Lemal. He then studied under Dennis
Dougherty at the California Institute of Technology as an NIH Predoc-
toral Fellow. After obtaining his PhD in 2005, he worked as an NIH Post-
doctoral Fellow at Yale University with Alanna Schepartz. He was
appointed as assistant professor in the Department of Chemistry at the
Thioamides are single-atom substitutions of the canoni-
cal amide bond in which the carbonyl oxygen is replaced by
a sulfur atom. This substitution lends thioamides distinct
physiochemical properties compared to their oxygen coun-
terparts (herein referred to as ‘oxoamides’ for clarity), while
being similar in size and structure.1 Thioamides exhibit in-
creased nucleophilicity and electrophilicity,2 greater metal-
binding affinity,3 altered hydrogen-bonding propensities,4
and unique spectroscopic features.5,6 These properties have
been exploited in numerous applications wherein thioam-
ide-containing peptides have been used as metal-binding
substrates,7 photoswitches,8 probes to monitor peptide or
protein folding,6,9 protease inhibitors,10 and fluorescence
quenchers.5,11,12 Improved methods for the synthesis of
University of Pennsylvania in 2008 and in the Biochemistry and Molecu-
lar Biophysics group in the Perelman School of Medicine in 2013. In
2015, he was promoted to the rank of associate professor. Prof. Peters-
son’s research focuses on the development of new methods for protein
labeling with synthetic amino acids and their application to studying
protein folding and stability. He has been the recipient of several
awards, including a Sloan Fellowship, an NSF CAREER award, recogni-
tion as a Searle Scholar, and a 2017 Thieme Chemistry Journals Award.
thioamide-containing peptides will enable such biophysical
experiments, as well as studies of thioamide-containing
natural products.13
For these and other types of applications, site-specific
incorporation of thioamides into peptides is required. This
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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D. M. Szantai-Kis et al.
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is usually achieved by synthesizing thioamide precursors
which can then be incorporated into peptides using fluo-
renylmethyloxycarbonyl (Fmoc)-based solid-phase peptide
synthesis (SPPS). The activated precursors can be synthe-
sized from Fmoc-protected amino acids in three steps
(Scheme 1) based on a nitrobenzyltriazolide (Nbt) route de-
veloped by Rapoport et al.14 Thioamides are generally com-
patible with the reagents used in SPPS including bases, acti-
vators, cleavage additives, and trifluoroacetic acid (TFA).
amides.21 However, since the half-life of the Fmoc group un-
der these conditions is 20–45 seconds, this approach sig-
nificantly decreases the yield of isolated protein.22
A 20% (v/v) solution of piperidine in dimethylforma-
mide (DMF) is the most commonly used solution for Fmoc
deprotection in SPPS, but there are alternatives: A 50% (v/v)
solution of morpholine in DMF is considered a milder con-
dition and is often used for sensitive glycopeptides.23 In
contrast, a 2% (v/v) solution of 1,8-diazabicyclo[5.4.0]un-
dec-7-ene (DBU) is considered a harsher deprotection con-
dition.24 Unlike piperidine or morpholine, DBU is a non-nu-
cleophilic base and is not able to scavenge the reactive
dibenzofulvene group resulting from the Fmoc deprotec-
tion.24 Therefore, either a nucleophile needs to be added as
a scavenger, or reaction times must be kept short and the
resin washed extensively after deprotection.
Here, we demonstrate that for the synthesis of thioam-
ide-containing peptides (herein referred to as ‘thiopep-
tides’) the use of 2% (v/v) DBU in DMF as an Fmoc-deprotec-
tion solution is superior compared to 20% (v/v) piperidine
in DMF. We synthesized several thiopeptides with either of
the aforementioned deprotection solutions. Throughout
this series, peptides synthesized with 2% DBU typically ex-
hibited a lower percentage of epimerization than those
synthesized with 20% piperidine, as well as higher isolated
yield. Additionally, we encountered the formation of a side
product when using piperidine or morpholine in the syn-
thesis of Nε-thioacetyl lysine (Lys(AcS)) containing peptides.
This side product was not separable from the desired prod-
uct via high-performance liquid chromatography (HPLC).
However, the use of 2% DBU avoided the formation of this
side product altogether. Thus, DBU deprotections are gen-
erally superior to piperidine deprotections for both back-
bone and side-chain thioamides.
Scheme 1 Synthesis of activated nitrobenzotriazolide (Nbt) thioamide
precursors for SPPS and their use to synthesize thioamide containing
peptides. Reagents and conditions: i) Isobutyl chloroformate (IBCF), 4-ni-
tro-o-phenylenediamine, N-methylmorpholine, THF, –20 °C → 25 °C,
Ar;15 ii) P4S10, Na2CO3, THF, Ar;16 iii) NaNO2, 95% AcOH(aq), 0 °C.17
There are three side reactions that can often occur with
thioamide-containing peptides. First, prolonged exposure
to high TFA concentrations during cleavage of the peptide
from the resin can lead to an Edman degradation type reac-
tion resulting in backbone cleavage at the n+1 position18
(see Supporting Information, Figure S2). Therefore, espe-
cially for longer peptides, one must consider a tradeoff be-
tween complete removal of acid-labile protecting groups
and degradation when determining the duration of TFA ex-
posure. Second, our laboratory as well as Chatterjee et al.
reported that thioamide precursors can react with residual
amounts of water during coupling, leading to the exchange
of sulfur for oxygen.12,19 The use of anhydrous CH2Cl2 great-
ly reduced the formation of the undesired oxoamide. Last,
epimerization can occur at the α-carbon of the thioamino
acid (pKa = ca. 13, see our rationale for estimating this value
in Supporting Information20). Prolonged exposure to base
during Fmoc deprotection can catalyze this epimerization
reaction. Recently, it was reported that lowering the con-
centration of piperidine and shortening Fmoc deprotection
times to one minute resulted in less epimerization of thio-
We designed a set of model peptides containing a mix-
ture of nonpolar (Phe and Ala) and polar (Lys and Glu) ami-
no acids (Table 1). For each sequence, 7-methoxycoumarin-
4-ylalanine (Mcm or μ) was installed as the first amino acid
on 2-chlorotrityl resin. This chromophore has an absorp-
tion maximum at 325 nm and allows for facile tracking of
the peptides by analytical HPLC. Thioamides also have a
shifted absorption maximum (272 nm) relative to oxoam-
ides (215 nm), which can be used to characterize the thio-
peptides. To discriminate between genuine L-thiaomino
acid peptides and their D-epimers, we synthesized both L-
and D-thionitrobenzotriazolide monomers (Fmoc-alaS-NBt
and Fmoc-pheS-NBt, using the lower-case letter convention
for D-amino acids) for inclusion into the model peptides.15–
17
The retention times for the authentic D-thioamino acid
peptides were used as standards for each of the first four
sets of thiopeptides. Additionally, a separate assay was per-
formed to assess how each condition affected yield. In these
experiments, 7-methoxycoumarin-4-acetic acid (Mca) was
added to each peptide sample to a final concentration of
500 μM, representing the maximum theoretical yield of
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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D. M. Szantai-Kis et al.
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peptide in each HPLC sample. The integration value of this
standard peak was compared to the integration of the prod-
uct peak in each chromatogram (at 325 nm) to calculate the
yield.
Table 1 Comparison of Yield and Epimerization between Peptides
Synthesized with either DBU or Piperidinea
We began our analysis with peptide 1, which contained
a thioamide linkage at the central Phe. Treatment with 20%
piperidine (v/v) in DMF for 20 minutes led to epimerization
of 16%, as calculated by the ratio of peak integrations (at
325 nm) for the L- and D-epimers. The identical masses of
the L- and D-product peaks were confirmed by matrix-as-
sisted laser desorption ionization mass spectrometry
(MALDI-MS). To test DBU deprotection conditions, the same
peptide was synthesized and elongated up to the thioamino
acid position using standard SPPS with 20% piperidine
deprotections. After the thioacylation reaction, the peptide
was treated with 2% v/v DBU in DMF (2 mL) for two minutes
before draining and washing the resin and then repeating
the process twice more. Each subsequent deprotection was
done using these conditions. It should be noted that a scav-
enger was not used in the deprotection solution, but no
characterizable byproducts containing the mass of a diben-
zofulvene adduct were observed. The aforementioned
deprotection conditions lead to only 3% epimerization, sig-
nificantly decreasing the amount of D-byproduct observed
in comparison to the piperidine deprotections. In addition,
the yield was increased from 30% to 36% when DBU was
used (Table 1). Oxoamide controls for the L- and D-peptides
were also synthesized using piperidine as the deprotection
reagent and no epimerization was observed for these pep-
tides (Figure S10).
Encouraged by these results, we synthesized four addi-
tional peptide series (2–5) with L- and D-thioamino acids.
Series 2 changes the sequence to put the PheS linkage at the
N-terminus of the peptide, allowing us to quantify the epi-
merization effects from a single deprotection reaction. Only
1% epimerization was detected with piperidine; epimeriza-
tion was undetectable with DBU conditions. This result in-
dicates that the initial deprotection step may not have a sig-
nificant effect and implies that the thioamino acid is vul-
nerable to epimerization at each subsequent deprotection
step. The epimerization of thiopeptides may also depend
upon local sequence or the identity of the thioamino acid.
Therefore, we synthesized peptides 3 and 4 with AlaS at
each Ala position within the same sequence as peptide 2.
We detected 3% epimerization with piperidine and 1% epi-
merization with DBU for peptide 3. This is in agreement
with the expected additive effects of multiple piperidine
treatments. Unfortunately, the L- and D-epimers of peptide
4 were not separable by analytical HPLC. This highlights the
need for Fmoc removal conditions that minimize epi-
merization of thioamino acid positions, as epimerization
byproducts may not always be chromatographically distin-
guishable from the desired product.
Peptideb
Epimerization Purity
(%)
(%)c
Yield
(%)
KAFSAKμ (1) – piperidine
KAFSAKμ (1) – DBU
16.1
56.2
29.9
35.9
44.3
51.3
44.0
44.7
18.2
22.7
n/dd
n/dd
n/dd
n/dd
5.0d
1.0
61.1
89.0
88.5
68.5
67.3
71.5
71.6
8.6
FSAKAKμ (2) – piperidine
FSAKAKμ (2) – DBU
0.0
FASKAKμ (3) – piperidine
FASKAKμ (3) – DBU
3.2
1.0
FAESAKμ (5) – piperidine
FAESAKμ (5) – DBU
5.6
1.0
CVNYSEEFVQMMTAK (6) – piperidine
CVNYSEEFVQMMTAK (6) – DBU
CVNYEEFSVQMMTAK (7) – piperidine
CVNYEEFSVQMMTAK (7) – DBU
49.9
9.3
12.3
25.0
22.0e
28.1
4.5e
a Graph: Example chromatogram for epimer quantification and yield deter-
mination. L and D denote epimers of 1, KAFSAKμ and KAfSAKμ, respectively,
# denotes KAAKμ, the product of failed FS coupling. ^ denotes Mca stan-
dard used to quantify yield.
b Peptides in one-letter code. Superscript S denotes thioamino acids; μ =
7-methoxycoumarin-4-yl-alanine.
c Purity calculation based on HPLC peak area of desired product.
d No external standard added for yield quantification.
e Average of two experiments.
The peptides synthesized thus far contained nonpolar
thioamino acids, therefore we synthesized peptide 5 con-
taining a GluS linkage to verify that epimerization can be
suppressed independent of side-chain identity (although %
epimerization may vary depending on amino acid identity).
In agreement with the effects discussed in peptides 1–3,
DBU treatments for peptide 5 decreased epimerization
from 6% to 1%. The peak-area percentage was calculated for
peptides 1–3 and 5 in order to quantify the effects of DBU
deprotections on the purity of the products. In each case,
the purity of the desired L-peptide was similar across meth-
ods, with the exception of peptide 1, where purity was in-
creased using the DBU method (Figure S2). In samples con-
taining the Mca standard discussed above, the yield of de-
sired L-product was either maintained or increased through
use of DBU, with the most significant increases in yield ob-
served in peptides 1 and 2 (30% to 36% and 44% to 51%, re-
spectively). Taken together, these results indicate that in se-
quences where there are several deprotections after the
thioamide installation, it is beneficial to use DBU as a
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D
D. M. Szantai-Kis et al.
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deprotection method to reduce epimerization and even in-
crease yield and purity of the desired L-thiopeptide prod-
uct.
dine. Thus, we find that although the two methods give
peptides of comparable overall purity, the lower levels of
epimerization seen with DBU represent a significant advan-
tage for our method, as peptide epimers may not always be
separable by HPLC (as seen for peptide 4).
To extend these results to a longer, more sequence di-
verse system, we synthesized a peptide derived from the C-
terminus of the calcium-binding protein calmodulin
(CaM).25 We have described the synthesis and characteriza-
tion of CaM135-148-Cys135 including many thioamide substi-
tutions recently,26 and we wished to see what improve-
ments could be made by using DBU deprotection condi-
tions. We established two target sites at N-terminal and
We are also interested in incorporating side-chain thio-
amides as probes for fluorescence-quenching studies or
protein interactions and as mimics of post-translational
modifications. Thus, we attempted to synthesize peptide 9
(Figure 1) containing the side-chain thioamide Lys(AcS).
However, during this synthesis, we encountered a +51 mass
product. This product was not observed while synthesizing
the corresponding oxopeptide 8 (Figure 1), which contained
Nε-(acetyl)-lysine. This implied that the modification must
have been part of the thioamide motif. To investigate this,
we tested several different cleavage solutions to cleave the
peptide from the resin (Table S1). However, all tested cleav-
age conditions still resulted in the +51 mass adduct, leading
us to believe that this side product was not formed during
peptide cleavage.
central positions within the 14-mer peptide (CaM135-148
-
Cys135TyrS and CaM135-148-Cys135PheS141, 6-L and 7-L, re-
138
spectively). Each peptide was synthesized on a 50 μmol
scale on 2-chlorotrityl resin with either piperidine or DBU
deprotection conditions. Thiopeptide products were de-
tected by their characteristic absorbance at 272 nm and
percent epimerization calculated for each peptide based on
the retention times of an authentic D-thioamino acid pep-
tide (CaM135-148-Cys135pheS141, 7-D) and MALDI-MS analysis
of the peaks from analytical HPLC chromatograms (6 and
7). In each of the CaM thiopeptides, the epimerization with
piperidine is more exaggerated than with any of the model
peptides discussed thus far (50% and 28% for 6 and 7, re-
spectively). As expected, DBU deprotection decreased the
observed epimerization percentages for peptides 6 and 7 to
9% and 5%, respectively. These results highlight the posi-
tional and sequence variability in the amount of epimeriza-
tion observed with piperidine-based Fmoc removal, as well
as the net positive effects that DBU deprotections can have
in all cases observed here. However, by chromatographic
peak area percentage analysis, DBU does appear to decrease
the overall purity in 7 as it may lead to additional byprod-
ucts that we were unable to assign by MALDI-MS analysis.
We hope to further improve our methodology by using an
additive during Fmoc deprotection in order to decrease the
formation of undesired byproducts. Nevertheless, DBU
deprotections did lead to a 7% increase in peak area per-
centage for 6.
Figure 1 Lys(AcS)-containing peptides used to study byproduct forma-
tion under piperidine deprotection conditions. MALDI-MS masses of 8,
9 (using DBU deprotection), 10 (byproduct of 9 synthesis using piperi-
dine deprotection), and 11 (byproduct of 9 synthesis using morpholine
deprotection); Pip. = piperidine, Mor. = morpholine.
Finally, we compared our DBU protocol to the protocol
used in Mukherjee et al., which also reported reduced epi-
merization during thiopeptide synthesis.21 Peptide 1 was
We hypothesized that the +51 mass corresponded to
piperidine adduct 10. Indeed, thioamides have been used
previously to prepare amidines, as in Boger’s syntheses of
vancomycin derivatives.27 This was confirmed with several
experiments. We synthesized peptide 9 using either 50%
(v/v) morpholine in DMF or 2% (v/v) DBU in DMF as depro-
tection solutions. We observed a +53 mass when Fmoc
deprotections were performed with morpholine, which we
believe corresponds to peptide 11. No side product was ob-
served in the peptide synthesized with DBU. We also syn-
thesized peptide 12 (Figure 1), where Nα-(Boc)-Nε-(thioace-
tyl)lysine [Boc-Lys(AcS)] was installed at the N-terminus of
the peptide and no +51 side product was observed after
cleavage (TFA only to cleave Boc group, no base required).
synthesized up to the coupling of PheS , and then the resin
3
was split. For one half of the resin, the peptide was elongat-
ed using 2 × 1 minute Fmoc deprotections with 10% piperi-
dine, as in Mukherjee et al. For the other half of the resin,
we elongated the peptide using our procedure of 3 × 2 min-
ute deprotections with 2% DBU. We observed similar purity
(63% vs. 61% with DBU), but a higher degree of epimeriza-
tion (7% vs. 1% with DBU) in the synthesis using 10% piperi-
dine. We also synthesized the longer peptide 7 using the
same split-resin approach for direct comparison of the two
methods. Here, we observed somewhat lower purity (14%
vs. 17% with DBU) with significantly higher epimerization
(23% vs. 2% with DBU) in the synthesis using 10% piperi-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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D. M. Szantai-Kis et al.
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To further verify adduct formation, we dissolved Boc-
Lys(AcS) in 50% (v/v) piperidine in DMF and isolated lysyl
amidine 15 by HPLC (Scheme 2).28,29 NMR, UV/vis spectros-
copy, and high-resolution mass spectrometry confirmed
the identity of 15. Specifically, we observed cross peaks in a
nuclear Overhauser effect spectroscopy (NOESY) experi-
ment between the CH3 group on the side chain with the
piperidinyl ring as well as the protons attached to the δ-
carbon of the Lys side chain. Additionally, we observed cor-
relations of that same methyl group with two nitrogen at-
oms using proton–nitrogen heteronuclear multibond cor-
relation (HMBC) spectroscopy. A more detailed illustration
of the observed correlations can be found in the Supporting
Information (Figure S7). These amino acid and peptide
studies confirm the necessity of using DBU in Fmoc depro-
tections when synthesizing Lys(AcS)-containing peptides.
We are currently investigating the syntheses of peptides
containing other side-chain thioamide groups.
Funding Information
This work was supported by funding from the National Science Foun-
dation (NSF CHE-1150351 to E.J.P.). Instruments supported by the
NSF and National Institutes of Health include: HRMS (NIH RR-
023444) and MALDI-TOF MS (NSF MRI-0820996). C.R.W. thanks the
NIH for funding through the Structural Biology and Molecular Bio-
physics Training Program (T32 GM008275). T.M.B. thanks the NIH for
funding through the Chemistry-Biology Interface Training Program
(T32 GM071399).
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Acknowledgment
We thank Dr. George Furst and Dr. Jun Gu for assistance with NMR
spectroscopy and Dr. Charles Ross III for assistance with HRMS.
Supporting Information
Supporting information for this article is available online at
https://doi.org /10.1055/s-0036-1589027. A detailed description of
materials and experimental methods used in this manuscript can be
found in the Supporting Information.
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References and Notes
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747.
(2) (a) Wipf, P.; Hayes, G. B. Tetrahedron 1998, 54, 6987.
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Scheme 2 Synthesis of 15. Reagents and conditions: i) ethyl dithioace-
tate, Na2CO3, EtOH–H2O;28 ii) 50% piperidine in DMF.29
With the preparation of each backbone thiopeptide sys-
tem, we have demonstrated reduction of epimerization at
the thioamino acid α-carbon by performing Fmoc-depro-
tection reactions with DBU in the place of piperidine. Al-
though only four different amino acids were tested (AlaS,
PheS, GluS, and TyrS), the positional and sequence variability
with which this effect is demonstrated provides strong evi-
dence that this result is general. Furthermore, we demon-
strated that in at least one side-chain thioamide case, the
use of DBU as the deprotection reagent was superior to
piperidine or morpholine. While we have not yet observed
a piperidine adduct in a backbone thioamide, it may occur
at sterically accessible sites such as GlyS residues. Such ad-
ducts would presumably not be stable, but could lead to
Edman-type degradation, as well as hydrolysis of the pep-
tide backbone or oxoamide formation by reaction with wa-
ter under the acidic resin cleavage conditions. All of these
side reactions would be avoided by using DBU for Fmoc
deprotections during peptide synthesis. We expect that
these synthetic developments will help to improve the ac-
cessibility of thioamide-containing peptides and proteins,
further enabling the broad spectrum of biophysical and
biochemistry experiments made possible by this versatile
functional group.
(15) Thioamino acid precursors for SPPS were synthesized as Fmoc-
thionitrobenzotriazolide monomers. The synthesis follows a
standard route shown in Scheme 1. The synthesis of Fmoc-PheS-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

F
D. M. Szantai-Kis et al.
Letter
Synlett
Nbt serves as an example. See the Supporting Information for
structures of compounds S1–S3.
(19) Mukherjee, S.; Verma, H.; Chatterjee, J. Org. Lett. 2015, 17, 3150.
(20) (a) Bordwell, F. G.; Bartmess, J. E.; Hautala, J. A. J. Org. Chem.
1978, 43, 3095. (b) Bordwell, F. G.; Fried, H. E. J. Org. Chem. 1991,
56, 4218. (c) Bordwell, F. G.; Ji, G. Z. J. Am. Chem. Soc. 1991, 113,
8398. (d) Huang, Y.; Jahreis, G.; Lucke, C.; Wildemann, D.;
Fischer, G. J. Am. Chem. Soc. 2010, 132, 7578. (e) Stroud, E. D.;
Fife, D. J.; Smith, G. G. J. Org. Chem. 1983, 48, 5368.
(21) Mukherjee, S.; Chatterjee, J. J. Pept. Sci. 2016, 22, 664.
(22) Zinieris, N.; Leondiadis, L.; Ferderigos, N. J. Comb. Chem. 2005, 7,
4.
(9H-fluoren-9-yl)methyl(R)-{1-[(2-amino-5-nitrophe-
nyl)amino]-1-oxo-3-phenylpropan-2-yl}carbamate (S1)
Fmoc-Phe-OH (1.29 mmol, 500 mg) was dissolved in THF (15
mL) and stirred under argon. The solution was then cooled to
–10 °C. NMM (2.58 mmol, 0.284 mL) was slowly added. Next,
isobutyl chloroformate (1.42 mmol, 0.186 mL) was carefully
added dropwise. The reaction was then stirred at –10 °C for 15
min, after which 4-nitro-o-phenylenediamine (1.42 mmol, 218
mg) was added. The reaction was stirred at –10 °C for 2 h and
continued stirring at r.t. overnight under argon. The next day
the solvent was removed in vacuo. The yellow solid was dis-
solved in DMF (20 mL), and the product was precipitated by
addition of sat. KCl solution (100 mL). This precipitate was fil-
tered and washed with cold water and Et2O. The product was
dried under vacuum overnight. Compound S1 (1.13 mmol, 589
mg) was obtained in a crude yield of 87.4% and used without
further purification
(23) Liebe, B.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1997, 36, 618.
(24) Wade, J. D.; Bedford, J.; Sheppard, R. C.; Tregear, G. W. Pept. Res.
1991, 4, 194.
(25) Chin, D.; Means, A. R. Trends Cell Biol. 2000, 10, 322.
(26) Walters, C. R.; Szantai-Kis, D. M.; Zhang, Y. T.; Reinert, Z.; Horne,
W. S.; Chenoweth, D. M.; Petersson, E. J. Chem. Sci. 2017, 8, 2868.
(27) Okano, A.; James, R. C.; Pierce, J. G.; Xie, J.; Boger, D. L. J. Am.
Chem. Soc. 2012, 134, 8790.
(28) N2-(tert-Butoxycarbonyl)-N6-ethanethioyl-L-lysine
(Boc-
(16) (9H-Fluoren-9-yl)methyl-(R)-(1-[(2-amino-5-nitrophe-
nyl)amino]-3-phenyl-1-thioxopropan-2-yl)carbamate (S2)
Na2CO3 (1.13 mmol, 120 mg) and P4S10 (1.13 mmol, 502 mg)
were suspended in THF (20 mL) and was stirred under argon
atmosphere at r.t. for 30 min. The reaction was cooled to 0 °C.
Compound S1 (1.13 mmol, 589 mg) was dissolved in THF (10
mL) and added to the reaction. After 1.5 h, the reaction was
complete and condensed to dark yellow oil by rotary evapora-
tion. The crude product was run over a short silica plug column
to remove insoluble P4S10 aggregates. The crude product was
purified by flash using EtOAc–hexanes (1:1). The desired
product S2 was obtained as an orange powder in 54.2% yield
(0.612 mmol, 330 mg). 1H NMR (500 MHz, CDCl3): δ = 9.07 (s, 1
H), 7.90 (dd, J = 9.0, 2.6 Hz, 1 H), 7.73 (dd, J = 10.6, 7.5 Hz, 2 H),
7.58 (s, 1 H), 7.48 (d, J = 7.5 Hz, 1 H), 7.41 – 7.32 (m, 3 H), 7.30–
7.17 (m, 9 H), 6.48 (d, J = 9.0 Hz, 1 H), 5.78 (s, 1 H), 4.86 (s, 1 H),
4.10 (dt, J = 16.7, 8.3 Hz, 5 H), 3.21 (d, J = 9.2 Hz, 0 H), 1.59 (s, 1
H), 1.36–1.04 (m, 1 H).
Lys(AcS)-OH, 14)
Boc-Lys-OH (13, 369 mg, 1.50 mmol, 1.0 equiv) was suspended
in EtOH (4.4 mL) and 10% (w/v) Na2CO3 solution (4.0 mL) added.
Ethyl dithioacetate (189 μL, 1.65 mmol, 1.1 equiv) added and
stirred overnight. The solvent was removed in vacuo and the
solid redissolved in H2O (10 mL). The reaction mixture was
acidified with 3 M HCl until the solution became milky white
(ca. pH 2). The aqueous phase was extracted three times with
CHCl3 (10 mL). The combined organic phase was dried over
Na2SO4, and the solvent was removed in vacuo. The product was
obtained as yellow foam in high yield (423 mg, 1.39 mmol,
1
92.4%). H NMR (500 MHz, CDCl3): δ = 10.88 (s, 1 H), 8.37 (s, 1
H), 5.35 (d, J = 7.7 Hz, 1 H), 4.07 (d, J = 75.4 Hz, 1 H), 3.52 (s, 2 H),
2.50–2.34 (m, 3 H), 1.77 (s, 1 H), 1.69–1.49 (m, 3 H), 1.42–1.23
(m, 11 H). ESI+-HRMS: m/z calcd for C13H25N2O4S+: 305.1535;
found [M + H]+: 305.1556.
(29) N2-(tert-Butoxycarbonyl)-N6-[1-(piperidin-1-yl)ethylidene]-
L-lysine (15)
(17) (9H-Fluoren-9-yl)methyl-(R)-(1-{6-nitro-1H-
Boc-Lys(AcS)-OH (14, 133 mg, 0.438 mmol) was dissolved in
50% (v/v) piperidine in DMF (2 mL). After stirring for 5 h, the
reaction mixture was diluted with 0.1% TFA in H2O and purified
by reverse phase HPLC. Fractions containing product 15 or
unreacted starting material 14 were collected separately and
lyophilized. After lyophilization starting material 14 was dis-
solved in 50% (v/v) piperidine in DMF and, after 5 h the reaction
was purified as before. This procedure was repeated one more
time until enough product was collected for NMR analysis (1.90
mg, 4.05 μmol, 0.9%). 1H NMR (500 MHz, DMSO-d6): δ = 12.58
(s, 1 H), 8.68 (s, 1 H), 7.05 (d, J = 7.8 Hz, 1 H), 3.84 (td, J = 8.8, 4.6
Hz, 1 H), 3.57 (s, 4 H), 3.32 (s, 2 H), 2.28 (s, 3 H), 1.67–1.54 (m, 8
H), 1.49 (dt, J = 13.3, 7.0 Hz, 2 H), 1.37 (s, 9 H), 1.36–1.28 (m, 2
H). 13C NMR (126 MHz, DMSO): δ = 174.28, 162.44, 155.65,
78.02, 53.36 (+), 49.45 (–), 46.48 (–), 43.89 (–), 30.38 (–), 28.86
(–), 28.25 (+), 25.64 (–), 24.74 (–), 23.06 (–), 22.65 (–), 14.35 (+).
ESI+-HRMS: m/z calcd for C23H27N2O4S+: 356.2549; found [M +
H]+: 356.2558. Additional 2D NMR correlations are given in the
Supporting Information.
benzo[d][1,2,3]triazol-1-yl}-3-phenyl-1-thioxopropan-2-
yl)carbamate (S3)
Compound S2 (0.612 mmol, 330 mg) was dissolved in 95%
AcOH(aq) (10 mL) and cooled to 0 °C. After 5 min, NaNO2 (0.765
mmol, 52.8 mg) was added slowly to the reaction. After 30 min,
ice-cold Milli-Q water (100 mL) was added to the reaction. The
resulting orange precipitate was filtered and washed with addi-
tional cold Milli-Q water. After drying the product was obtained
as an orange powder in 84.0% yield (0.514 mmol, 256 mg) and
was used directly in SPPS without further purification. 1H NMR
(500 MHz, CDCl3, major rotamer only): δ = 9.61 (s, 1 H), 8.44 (d,
J = 8.9 Hz, 1 H), 8.29 (d, J = 8.9 Hz, 1 H), 7.74 (d, J = 7.6 Hz, 2 H),
7.52 (t, J = 6.9 Hz, 2 H), 7.38 (t, J = 7.6 Hz, 2 H), 7.28 (q, J = 8.0 Hz,
2 H), 7.23–7.13 (m, 5 H), 6.56 (d, J = 7.4 Hz, 1 H), 5.66 (d, J = 9.3
Hz, 1 H), 4.46–4.35 (m, 1 H), 4.33 (t, J = 9.0 Hz, 1 H), 3.40 (dd, J =
14.0, 5.4 Hz, 1 H), 3.10 (dd, J = 13.8, 8.0 Hz, 1 H). ESI+-HRMS: m/z
calcd for C30H23N5O4S+: 550.1549; found [M + H]+: 550.1550.
(18) Stolowitz, M. L.; Paape, B. A.; Dixit, V. M. Anal. Biochem. 1989,
181, 113.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F