A Bottom-Up Approach to Preserve Thioamide Residue Stereochemistry during Fmoc Solid-Phase Peptide Synthesis

Article abstract of DOI:10.1021/acs.orglett.9b02598

Thioamides are useful biophysical probes for the study of peptide structure and folding. The α-C stereochemistry of thioamide amino acids, however, is easily epimerized during solid-phase peptide synthesis (SPPS), which limits the sequence space that is a

Full text of DOI:10.1021/acs.orglett.9b02598

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
A Bottom-Up Approach To Preserve Thioamide Residue
Stereochemistry during Fmoc Solid-Phase Peptide Synthesis
Luis A. Camacho, III, Bryan J. Lampkin, and Brett VanVeller*
Department of Chemistry, Iowa State University, Ames, Iowa 50011, United States
*
S Supporting Information
ABSTRACT: Thioamides are useful biophysical probes for the study of
peptide structure and folding. The α-C stereochemistry of thioamide amino
acids, however, is easily epimerized during solid-phase peptide synthesis
(
SPPS), which limits the sequence space that is available to thioamide
incorporation. This work demonstrates that the α-C stereochemistry of
thioamides can be reversibly protected in a manner that is compatible with the
standard methodology of SPPS to enable the facile implementation of
thioamide probes.
hioamides are versatile chemical probes for studying the
function, behavior, and stability of protein structure.
the development of improved methods for thioamide
synthesis.
1
,2
1b
T
Because thioamides display different strengths of hydrogen-
bond-donating and -accepting ability relative to amides (1),
they have been used to probe the role of hydrogen bonding in
The goal of this study was to develop a general strategy for
the robust synthesis of thioamide-containing peptides with
high stereochemical integrity.
3
−5
protein secondary structure. Alternatively, the greater Lewis
basicity of sulfur in thioamides (2) has been used to
interrogate possible n→π* interactions between adjacent
peptide bonds (i to i + 1).
employed in photochemical schemes as photoswitches or as
fluorescent quenchers to study conformational changes in
peptides.
In view of the importance of thioamides in peptide
chemistry, methods for their incorporation into peptides
with robust stereochemical integrity are highly desirable.
Activated thioamide precursors can be prepared from Fmoc-
The acidity of the α-proton next to a thioamide (2) moiety
is ∼3 pK units lower than that of the corresponding oxoamide
a
20,24
(
1) (Figure 1).
This difference arises from the ability of
6
−8
Thioamides can also be
the lower-energy π*(CS) antibonding orbital relative to the
π*(CO) counterpart to more favorably stabilize the anion of
the conjugate base through resonance. Thus, we reasoned that
raising the energy of the orbital responsible for stabilizing the
9
,10
1
1−15
1
6
17,18
protected amino acids
and introduced into peptides
following standard solid-phase peptide synthesis (SPPS)
protocols. While this approach provides for site-selective
insertion of a thioamide into the sequence, the α-C of the
thioamide residue is prone to racemization under conditions
19−22
required for Fmoc deprotection.
Subsequent coupling and
Fmoc deprotection of additional residues following the
thioamide leads to further deterioration of the stereochemical
integrity of the thioamide residuein addition to other side
20−22
reactions that can compromise the yield.
Indeed, the lack
of literature precedent for a C-terminal thioamide, we surmise,
is due to its current synthetic inaccessibility, where a thioamide
at the C-terminus is necessarily exposed to Fmoc SPPS
23
reagents for longer periods. Accordingly, thioamides are
often incorporated only within a few residues of the end of the
sequence to limit such side reactivity. This approach, however,
leaves a considerable amount of sequence space potentially off-
limits to these important biophysical probes. Moreover, the
growth in the number of thioamide-containing natural
products, which continue to be discovered, further motivates
Figure 1. Rationale for the acidities of the α-CH moieties in 1−3.
Orbital energies were estimated by DFT B3LYP/6-311+g(d,p) NBO
analysis.
Received: July 24, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02598
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
conjugate base would confer resistance to epimerization at the
α-C stereocenter. We hypothesized that converting the
thioamide into a thioimidate (3) would present a higher-
energy π*(CN) orbital that does not stabilize the conjugate
base as well and, in turn, decreases the acidity of the α-CH
moiety.
reasonable to conclude that significant epimerization of the
thioamide α-C would occur if peptide elongation were carried
out beyond even a few couplings after coupling of the
thioamide residue. Moreover, such stereochemical impurities
may be impossible to separate or even detect with longer
sequences. While modified conditions for Fmoc deprotection
with abbreviated deprotection times and lower concentrations
The protection of a thioamide as a thioimidate has been
employed previously in the synthesis of natural product
of the base have been reported in an attempt to reduce the
2
5
20,21
derivatives, but the α-CH pK has not been determined.
epimerization reaction shown here,
a drawback of shorter
a
Thus, to test our hypothesis, we synthesized thioimidate 5
from thioamide 4 (Scheme 1) to assess how 5 might resist
epimerization.
and more dilute Fmoc deprotection conditions is the potential
for incomplete deprotection, leading to amino acid deletions.
Ultimately, the results in Figure 2 suggest that any attempts to
modify the conditions will be stymied by the fact that the α-
CH of the thioamide residue is simply too acidic to tolerate the
basic conditions of Fmoc SPPS.
27
Scheme 1. Protection of a Thioamide as a Thioimidate
In contrast to thioamide 4, the thioimidate protection in 5
withstood epimerization to a far greater extent in the presence
of 20% piperidine in DMF (Figure 2). Alternatively, because 5
epimerizes at similar rates as 4 in the presence of DBU, it
appears that the thioimidate is acidic enough to be
deprotonated by bases that are stronger than piperidine
We chose to investigate the stability of the model dimer
(
pKaH = 11). The significantly diminished rate of epimerization
(
X)
Cbz-Phe -Ala-OMe (where X = S or SMe) because
phenylalanine thioamides have been shown to be especially
of 5 over 300 min led us to conclude that the pK of the
a
thioimidate must be greater than those of thioamides (>12)
but ultimately lower than those of amides (<15) (Figure 1).
While the rate of epimerization is likely sequence-specific, the
results in Figure 2 confirm our design hypothesis that
thioimidates will be far more resistant to epimerization than
their thioamide analogues during SPPS. In addition, the
higher-energy π*(CN) orbital is less electrophilic, protect-
ing the thioamide site against nucleophilic degradation, which
is a possible side reaction of thioamides during lengthy SPPS
procedures. This result represents a significant advancement
over the current status quo for thioamide stability during
synthesis.
20
prone to epimerization at the α-C. Notably, the conditions to
make 5 are compatible with SPPS techniques (MeI/DIEA)
and could conceivably be applied directly on resin following
installation of the thioamide using standard thioacylating
1
7,18
agents.
We next exposed each dimer (4 and 5) to bases that are
commonly employed during the Fmoc deprotection phase of
SPPS and monitored the extent of epimerization by LCMS
(
Figure 2). Both dimers were also tested against trialkylamine
We next sought to evaluate the sensitivity of thioimidates to
acidic conditions that are commonly used to cleave the peptide
from the supporting solid resin and/or to cleave side-chain
protecting groups (Figure 3). We observed that thioimidate
dimer 5 decomposed rapidly within minutes upon exposure to
even dilute (2%) TFA. Alternatively, 5 displayed excellent
stability toward strong proton-donor solvents like TFE and
Figure 2. Epimeric stability of 4 and 5 with typical reagents used in
SPPS. Solutions of 4 and 5 (0.1 M) in DMF with the indicated bases
(
v/v) relative to 1,3,5-trimethoxybenzene as internal standard.
bases commonly used for the peptide coupling steps (e.g.,
DIEA and NMM). Both 4 and 5 showed no epimerization with
DIEA or NMM over the times measured (Figure S1), which
20,26
agrees with previous observations.
The thioamide dimer 4 showed significant epimerization in
under an hour in the presence of piperidine and DBU.
Traditional time frames for Fmoc deprotection using 20%
piperidine can be as long as 10 min per coupling. Thus, it is
Figure 3. Stability of 5 (0.1 M) with typical solvents/reagents used
for peptide cleavage from SPPS resin.
B
DOI: 10.1021/acs.orglett.9b02598
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
HFIP that are employed to cleave protected peptides from so-
called super-acid-sensitive resins (e.g., 2-chlorotrityl resin).
The sensitivity of thioimidates to acid was not unexpected
given the known basicity of imidatesthe oxygen congeners of
This sensitivity, however, must
Table 1. Protection of α-C Stereochemistry during SPPS
S
thioimidate
protection
epimerization of F
a
peptide
sequence
(%)
S
28−30
6
8
9
0
11
Fmoc-F AKFG-OH
−
<0.01
12.8
<0.01
22.1
3
(imidate pK ∼ 6−7).
aH
S
H-KAF AKFG-OH
no
yes
no
yes
be taken into account during synthetic planning. Thioamides
are also prone to side reactivity during TFA cleavage from the
resin, but strategies to mitigate unwanted reactivity have been
S
H-KAF AKFG-OH
S
1
H-AAKAF AKFG-OH
H-AAKAF AKFG-OH
S
1
4,26,31
<0.01
reported.
Thus, we propose that the thioimidate must be
a
Determined from peak areas of HPLC traces and control peptides
converted back to a thioamide prior to any treatment with
TFA. Alternatively, the peptide containing the thioimidate can
be cleaved intact from fast-cleaving resins (e.g., 2-chlorotrityl
resin) with TFE and HFIP solutions. Thus, thioimidate
protection functions in analogy to the protecting groups of the
sensitive functional groups of amino acid side chains during
SPPS, in which fully protected peptides can be cleaved under
similar conditions of TFE or HFIP solutions. With this proof-
of-concept reactivity established, we next sought to demon-
strate conditions for the interconversion between thioamide
and thioimidate that could be easily implemented within the
SPPS workflow.
s
containing authentic D-F . Identity confirmed by MALDI-TOF.
residue occurs during subsequent Fmoc deprotection of
additional amino acids. Alternatively, peptides 9 and 11 were
synthesized by immediately protecting the thioamide as a
thioimidate after installation and subsequently converting the
thioimidate back to the thioamide once the final sequence was
complete. In this manner, epimerization of the peptide was
curtailed.
Because thioamides can be introduced into sequences using
17,18
reliable thioacylating reagents,
there is a perception that
current methods to incorporate thioamides into peptides via
SPPS are sufficient. However, that perception is belied by the
poor stability that thioamides display once they are
incorporated and subsequent amino acids are coupled onto
the peptide. This drawback may explain the limited sequence
space in which these important biophysical probes have been
employed.
To assess the viability of thioimidate protection during
SPPS, we synthesized the short sequence F AKFG (6) on 2-
(
S)
chlorotrityl resin following established procedures for the
26
introduction of thioamide residues on-resin. The thioamide
was subsequently protected as the thioimidate with MeI and
DIEA in DMF, conditions that are compatible with SPPS
32
resins (6 → 7; Figure 4). After installation of the thioimidate
We have developed a strategy to reversibly protect the
thioamide as a thioimidate, which dramatically reduces
epimerization of the α-C stereocenter. Our strategy for
thioamides operationally mimics the current use of protecting
groups for the sensitive functional groups of amino acid side
chains and can be easily implemented within current Fmoc
SPPS workflows. This work therefore provides immediate and
enabling tools that allow peptide chemistry laboratories to
address questions in the folding and stability of proteins by
opening up previously uncharted sequence space to the
incorporation of thioamide biophysical probes.
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b02598.
Experimental details, NMR spectra, and LC traces
Figure 4. On-resin interconversion of thioamide to thioimidate and
back to thioamide. The reactions can be performed using a standard
peptide synthesis reaction vessel and were tracked with UPLC and
MALDI-TOF.
(PDF)
AUTHOR INFORMATION
Corresponding Author
bvv@iastate.edu
■
*
protection, SPPS could continue normally from 7 with Fmoc
deprotection and amino acid coupling cycles to elongate the
peptide. At the conclusion of the sequence, conversion back to
ORCID
Bryan J. Lampkin: 0000-0002-8633-1227
Brett VanVeller: 0000-0002-3792-0308
Notes
the thioamide can then be accomplished with H S gas in the
2
presence of a weak base such as collidine (7 → 6; Figure 4).
The procedure for protection and deprotection of the
thioamide described in Figure 4 can be easily implemented
within the standard SPPS workflow to improve the stereo-
chemical purity of thioamide-containing peptides (Table 1).
The synthesis of 8 and 10 was carried out using conventional
thioamide amino acid incorporation and peptide elongation.
Unsurprisingly, significant epimerization of the thioamide
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is dedicated to Prof. Ronald T. Raines on the
occasion of his 60-ish birthday. The authors thank the National
Science Foundation (Grant 1848261) and the Donors of the
C
DOI: 10.1021/acs.orglett.9b02598
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
American Chemical Society Petroleum Research Fund
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Org. Lett. XXXX, XXX, XXX−XXX