Cysteine pseudoprolines for thiol protection and peptide macrocyclization enhancement in fmoc-based solid-phase peptide synthesis

Article abstract of DOI:10.1021/ol5004725

Contrary to other studies, here we describe cysteine (Cys) pseudoproline-containing peptides with short deprotection times in TFA. The deprotection times fell in the same range as other protecting groups commonly used in SPPS (e.g., 1-3 h). Moreover, when using Cys pseudoprolines as peptide macrocyclization-enhancing moieties a considerable reduction in reaction time was observed compared to a peptide containing trityl protected Cys.

Full text of DOI:10.1021/ol5004725

Letter
pubs.acs.org/OrgLett
Terms of Use
Cysteine Pseudoprolines for Thiol Protection and Peptide
Macrocyclization Enhancement in Fmoc-Based Solid-Phase Peptide
Synthesis
Tobias M. Postma†,‡ and Fernando Albericio*,†,‡,§,∥
†Institute for Research in Biomedicine, 08028, Barcelona, Spain
‡CIBER-BBN, 08028, Barcelona, Spain
§Department of Organic Chemistry, University of Barcelona, 08028, Barcelona, Spain
∥School of Chemistry and Physics, University of KwaZulu Natal, 4001, Durban, South Africa
S
* Supporting Information
ABSTRACT: Contrary to other studies, here we describe
cysteine (Cys) pseudoproline-containing peptides with short
deprotection times in TFA. The deprotection times fell in the
same range as other protecting groups commonly used in
SPPS (e.g., 1−3 h). Moreover, when using Cys pseudoprolines
as peptide macrocyclization-enhancing moieties a considerable
reduction in reaction time was observed compared to a peptide containing trityl protected Cys.
ince the introduction of solid-phase peptide synthesis
S(SPPS) and its subsequent maturation through the
continuous refinement of reagents, linkers, resins, and
protocols, many complex peptides are readily accessible.1,2
SPPS now allows the preparation of complex peptides on a
large scale, thus facilitating the global commercialization of
complex peptide drugs, such as prialt and ziconotide.3,4 The
Figure 1. Structure of a pseudoproline (ψMe,Mepro) dipeptide.
prevalence of complex peptides is increasing, exemplified by the
fact that several multiple disulfide-containing peptides are
undergoing clinical trials.5 However, there are still many
obstacles to overcome in the synthesis of complex peptides,
such as addressing difficult sequences and orthogonal
protecting groups, and solving low coupling efficiencies and
solubility issues. In this regard, the development of novel tools,
protocols, and techniques in peptide synthesis are required to
tackle these difficulties. Developed by Mutter et al., pseudopro-
line dipeptide building blocks are a prime example of
innovation in complex peptide synthesis.6
Pseudoproline dipeptides have become powerful tools for the
synthesis of peptides containing difficult sequences.7 Mecha-
nistically, pseudoprolines act by disrupting the secondary
structure and increase the solubilization of protected peptides.
The disruption of the secondary structure, such as β-sheet
formation, is caused by the cisoid amide conformation of the
oxazolidine- or thiazolidine-based pseudoproline rings, which
introduce a “kink” in the backbone and disrupt backbone
hydrogen bonding analogous to Pro (Figure 1).8 The cisoid
pseudoproline conformation has also been utilized for reducing
aspartimide formation during Lansbury aspartylation.9−11
Oxazolidine-based dipeptides derived from Ser or Thr are
extensively used and commercially available as Fmoc-protected
dipeptides. Typically, the oxazolidine ring can be deprotected
to the parent amino acid within several hours using TFA based
cleavage cocktails, as illustrated in the synthesis of human
amylin.12
Conversely, thiazolidine based dipeptides have not gained
widespread use because of their high stability to TFA. In the
seminal paper by Mutter et al., deprotection times of 32 h were
reported for the removal of Cys pseudoprolines in linear
peptides to the corresponding deprotected Cys.6 This
observation was confirmed in a recent publication, where
treatment with TFA/TIS/H2O (95:2.5:2.5) for 36 h was
required to remove Cys pseudoproline in a linear peptide.13 In
addition, this publication claimed Cys pseudoproline removal in
head-to-tail cyclic peptides takes 13 days in TFA/TIS/H2O
(95:2.5:2.5) and requires the use of harsh acids, such as neat
trifluoromethanesulfonic acid, to achieve quantitative depro-
tection in a reasonable time frame. Furthermore, our group has
prepared head-to-tail cyclic peptides containing up to four Cys
pseudoprolines.14 In this case, we observed high stability to
TFA, thereby confirming the long deprotection times for
thiazolidine-based pseudoprolines in head-to-tail cyclic pep-
tides.
Received: February 13, 2014
Published: March 11, 2014
© 2014 American Chemical Society
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Pseudoprolines have been described as turn inducers.15 This
has been substantially developed by Jollifffe et al., which
focused mainly on Ser and Thr pseudoprolines as turn
inducers.16−18 In one study Cys pseudoprolines were used as
turn inducers.13 These studies noted a yield increase upon
macrocyclization; however, none of the previous studies
described the effect of pseudoprolines on macrocyclization
kinetics.
peptide revealed complete Cys pseudoproline removal (9) after
1 h of TFA treatment.
Scheme 2. Acidolysis of Peptide 3 (1 h in TFA/TIS/H2O
(95:2.5:2.5))
To widen the applicability of Cys pseudoprolines, we
initiated a study as part of our ongoing research into Cys
protection. Herein, we report on the remarkable Cys
pseudoproline lability in the same time scale as oxazolidine-
based pseudoprolines in a series of peptides. Moreover, we
identify a considerable increase in kinetics when using Cys
pseudoprolines as macrocyclization-enhancing moieties, which
was not observed in previous studies. In this regard, these
moieties significantly reduced the coupling time required for
complete peptide macrocyclization.
We first synthesized two random tetrapeptides using the
commercial Cys pseudoproline building blocks Fmoc-Ser(tBu)-
Cys(ψMe,Mepro)-OH and Fmoc-Ala-Cys(ψMe,Mepro)-OH
(Scheme 1). With the exception of Cys pseudoproline
Based on these results we prepared a small peptide library
with different sequences (Figure 2). For design considerations
Scheme 1. Acidolysis of Peptides 1 and 2 (1 h in TFA/TIS/
H2O (95:2.5:2.5))
Figure 2. Cys pseudoproline containing peptides.
in the library we used a series of nonapeptides, one octapeptide,
and one decapeptide in which we focused on diversifying the
N-terminal residues of the pseudoproline dipeptide building
bock, i.e., Xxx(PG)-Cys(ΨMe,Mepro).
The peptides were cleaved from the resin, and 2 h or less
were required for complete removal of the Cys pseudoprolines
at room temperature, except for peptides 23 and 26 which
required 45 °C for complete removal (Table 1). Peptide 23
Table 1. Deprotection Times of Cys Pseudoproline Peptides
dipeptides, standard protected Fmoc-amino acids were used
for all residues in peptide synthesis. Model peptides 2 and 5
were prepared on a Rink Amide resin using standard SPPS
protocols and were then cleaved from the resin using a 1 h
treatment with TFA/TIS/H2O (95:2.5:2.5). Chromatographic
and spectroscopic analysis of peptide 2 revealed an unusual
lability of the Cys pseudoproline to acidolysis. Only 65% of the
expected Cys pseudoproline was observed, and 35% of the
peptide was fully deprotected to the parent Cys (3). Analysis of
peptide 5 revealed a similar lability to acidolysis with 85% of the
Cys pseudoproline remaining after 1 h of TFA treatment.
Complete Cys pseudoproline removal in peptide 2 required
treatment with TFA/TIS/H2O (95:2.5:2.5) for 4 h while
peptide 5 required 6 h. These deprotection times are
significantly shorter than the previously reported deprotection
times of 32−36 h for Cys pseudoproline-containing linear
peptides.
peptide
Ψ pro
temp (°C) deprotection time (h) deprotection %
11
14
17
20
23
26
Ser-Cys
Ala-Cys
Asp-Cys
Asp-Cys
Lys-Cys
Lys-Cys
25
25
25
25
45
45
1
2
2
2
2
3
98
94
95
91
93
91
showed near-quantitative deprotecton whereas peptide 26 was
deprotected 57% in 3 h at rt and both peptides required 2−3 h
in TFA at 45 °C for complete removal. These results contrast
strongly with Cys pseudoproline deprotection times of 32−36
h, using the same cleavage cocktail, on different model
peptides.6,13
Our results strongly indicate that Cys pseudoproline removal
is sequence-dependent. The factors governing the Cys
pseudoproline deprotection times are not well understood.
This will be the focus of our ongoing research efforts, which
Peptide 7 which contained the Ser-Cys pseudoproline
sequence was prepared using standard SPPS and subsequently
cleaved from the resin (Scheme 2). Analysis of the cleaved
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Scheme 3. Macrocyclization Experiments of Peptides with and without Pseudoprolines
will be reported in due course. In addition, we noted that the
deprotection times fell in the same range as those for
oxazolidine pseudoprolines, thereby making Cys pseudopro-
lines more applicable as they do not require harsh acids for
deprotection.
peptides 41 and 46, which did not contain Cys pseudoprolines,
showed 52% and 70% macrocyclization, respectively. These
results show that the thiazolidine ring in Cys pseudoprolines
enhance macrocyclization by significantly decreasing the
coupling time.
Thiazolidine based Cys pseudoproline dipeptides have
previously been described to be highly stable to TFA and
require harsh acids for removal. As a result, these protecting
groups are not widely used. We observed markedly different
behavior to TFA, with deprotection times of 1 h for peptides 7
and 11 and several hours for the other examples. Our findings
show that Cys pseudoproline removal can be equally efficient as
that of the widely used oxazolidine pseudoprolines. Cys
pseudoprolines were shown to enhance the on-resin macro-
cyclization efficiency of two conotoxin derivatives by
significantly decreasing the coupling time required for
completion of the reaction. Thus, we envisage that the
combined use of Cys pseudoprolines and diphenylmethyl
(Dmp) protection will render a pair of Cys protecting groups
compatible with trimethoxyphenylthio (S-Tmp), trityl (Trt), or
phenylacetamido (Phacm) protecting groups, thus amplifying
the repertoire of Cys protection for the synthesis of complex
peptides. In conclusion, the short deprotection times and
enhanced macrocyclization efficiency of Cys pseudoprolines
significantly increase their applicability as Cys protecting
groups.
After observing that Cys pseudoprolines were removed using
short TFA treatments, we addressed the capacity of thiazolidine
rings to enhance peptide macrocyclization. This experiment
was based on the cisoid amide conformation of the thiazolidine
ring, through which the “kink” in the backbone would bring the
two reacting groups closer and consequently speed up the
macrocyclization. We chose two α-conotoxins (CnIB and A1.4)
from the ConoServer database that contained the Ser-Cys or
Ala-Cys sequences, which were used as suitable model systems
for Cys pseudoproline macrocyclization studies.19 In these
peptides two of the four Cys residues were replaced with allyl
protected Asp and a diaminopropionic acid (Dap) residue. The
experiment required four peptides, two conotoxin derivatives
with either a Ser-Cys or an Ala-Cys pseudoproline, and two
peptides with standard protected amino acids. The allyl
protecting groups were removed using palladium catalysis,
and the on-resin macrocyclization was performed using
diisopropylcarbodiimide (DIC) and Oxyma Pure as the
coupling system.20 After 2 h the coupling was stopped. A
negative Kaiser test showed the disappearance of amine in the
pseudoproline containing peptides, which indicated completion
of macrocyclization (Scheme 3, vials 1 and 3).21 This
observation contrasts with the incomplete macrocyclization
for the peptides that did not contain Cys pseudoprolines as
indicated by the presence of an amine in a positive Kaiser test
(Scheme 3, vials 2 and 4). Analysis of the peptides after
cleavage from the resin determined complete macrocyclization
of those containing Cys pseudoprolines (38 and 43), while
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures, characterization, spectro-
scopic and chromatographic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
■
*E-mail: fernando.albericio@irbbarcelona.org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Thomas Bruckdorfer and Mrs. Annick Vancolen
from Iris Biotech GmbH for providing us the Cys pseudopro-
line and for useful discussions. This work was partially
supported by the Marie Curie ITN MEMTIDE (FP7-
PEOPLE-ITN08), CICYT (CTQ2012-30930), and the Gen-
eralitat de Catalunya (2009SGR 1024).
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