Facile synthesis of sulfotyrosine-containing α-conotoxins

Article abstract of DOI:10.1039/d0ob01526a

α-Conotoxins (Ctx) can selectively target distinct subtypes of nicotinic acetylcholine receptors (nAChRs), which are closely related to a number of neurological diseases, and they have been considered as ideal probes and model peptide drugs. Sulfotyrosine

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DOI: 10.1039/D0OB01526A
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Facile synthesis of the sulfotyrosine-containing α-Conotoxins†
Changpeng Li, Chunmao He*
Received 00th January 20xx,
Accepted 00th January 20xx
α-Conotoxins (Ctx) can selectively target distinct subtypes of the nicotinic acetylcholine receptors (nAChRs), which are
closely related to a number of neurological diseases, and they have been considered as ideal probes and model peptide
drugs. Sulfotyrosine (sY) is an important post-translational modification and believed to modulate certain key protein-
protein interactions. Although sY modification has been indicated in several α-Ctx, its biological consequence has largely
remained unexplored, mostly because of the difficulties in both the extraction from biological samples and chemical
synthesis. Herein, we report a facile synthesis and folding strategy for obtaining the sY modified α-Ctx. This strategy is
based on the development of a simple and controlled deprotection of the neopentyl protecting group of the sulfate ester
as well as its compatibility with a step-wise oxidative folding of the two disulfide bonds. Eight sY modified α-Ctx peptides
were successfully synthesized in high purity and yield, and their serum stabilities were almost comparable with the non-
modified peptides.
DOI: 10.1039/x0xx00000x
synthetic advancements have made it possible to construct the
Introduction
sY modified peptides/proteins in
a highly controllable
fashion.13 Several acid-stable orthogonal protection groups of
the sY have been developed, which enables the direct
synthesis of sulfopeptides via standard Fmoc-SPPS.13-19 Among
them, the most widely used is the so-called cassette strategy
developed by Widlanski and co-workers, where the neopentyl
(nP) moiety is employed as a protection group in the monomer
Conotoxins (Ctx) belong to a large family of peptide toxins
derived from the venoms of cone snail, which act on a variety
of ion channels and receptors.1 α-conotoxin (α-Ctx) is a
subtype of Ctx, which typically has 12-19 amino acid residues
and two disulfide bonds (Cys1-Cys3 and Cys2-Cys4,
respectively), forming a rigid two-loop helical framework.2,3 α-
Ctx can selectively target ion channels and distinct subtypes of
the nicotinic acetylcholine receptors (nAChRs). Since nAChRs
are closely related with neurological diseases such as nicotine
addiction, severe pain, epilepsy, and Alzheimer's disease, α-Ctx
have been regarded as ideal molecular probes for target
validation and peptide drug development.4-6 The diverse
selectivity of α-Ctx has been largely attributed to the
comprising amino acid residues, where mutating key residues
can fine-tune their nAChR subtype specificity.7,8 Moreover, the
extracted native α-Ctx have been shown to have gone through
a number of post-translational modifications (PTM),7 especially
the tyrosine sulfation that leads to sulfotyrosine (sY)-
containing α-Ctx.9-11 While sY modification has been
considered a common and important mode of protein PTM,12
its biological impact on α-Ctx, be it the subtype specificity or
affinity, remains largely unexplored.
Fmoc-Tyr(OSO3-nP)-OH.16 As shown in Scheme
monomer can be conveniently installed in a peptide sequence,
after which the nP protection group can be removed by either
sodium azide or ammonium acetate treatment, at elevated
1, the
temperatures, e.g. 37–70 C. The resulting sulfopeptides can
then be purified by HPLC, typically using ammonium acetate
and acetonitrile (MeCN) as eluents. A number of sY containing
peptides/proteins have been successfully obtained with this
strategy, however, with the presence of free Cys in the peptide
sequence only in rare cases.20-24
The ammonium acetate solution used for both the nP
deprotection and HPLC purification is, in principle, not a buffer,
and may subject to pH changes when different solutes are
used.25 It can be imagined (also proved from this work, see
below) that free thiols, like in the case of α-Ctx, may be
susceptible to oxidation during the nP removal or the HPLC
purification, which complicates the disulfide pairing process.
As such, the current cassette strategy may not be compatible
with the synthesis of sY-modified peptides having multiple Cys
residues, like α-Ctx. As such, an alternative strategy will be
required, which is the aim of the present work.
It has been a general problem to produce and study the
sulfated peptides/proteins from biological systems due to the
lability of the sulfate ester group, fortunately, a number of key
School of Chemistry and Chemical Engineering; South China University of
Technology, Guangzhou 510640, China.
Email address: hecm@scut.edu.cn
†Electronic Supplementary Information (ESI) available: monomer and peptide
analytical data; table of conotoxin sequence and total synthetic yields. See
DOI: 10.1039/x0xx00000x
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purification were it to be used as the eluenVt,iewaAsrticilne Ontlhinee
literature.13 As such an alternative nP deprotection strategy is
DOI: 10.1039/D0OB01526A
needed in order to synthesize the sY-containing α-Ctx. During
the processing of peptide 1, we found that the nP group is not
stable if stored longer in aqueous solutions (data not shown).
It was also reported that the nP group can be removed after
native chemical ligation if left longer in the ligation buffer,21,22
we were interested to see whether the deprotection can just
happen in any aqueous (buffer) system. If this is the case, we
may achieve one-pot nP deprotection while folding of the α-
Ctx.
Scheme 1. Overview of the cassette strategy for the synthesis
of sulfopeptides using nP protecting group, highlighting the
major discoveries of the current work.
Results and discussion
Synthesis of non-sY modified conotoxin
We selected eight α-Ctx peptides, namely EpI, PnIA, PnIB, PnIC,
AnIA, AnIB, AuIA and PnMGMR-02, that have either been
reported or predicted (according to Sulfinator26) to be sY
modified, and their amino acid sequences are summarized in
Table S1. Initially, a direct synthetic strategy was employed,
where four Cys residues were left unprotected after SPPS.
Under a variety of folding conditions tested, multiple disulfide
isomers could always be observed (data not shown, which may
affect the overall yield) — a known major challenge in the
folding of these peptides.27 We therefore switched to a step-
wise disulfide pairing strategy as highlighted in Scheme 2 28
.
Here, the Cys residues 2 and 4 were side-chain protected with
Acm and the first disulfide bond was selectively formed
between Cys 1 and 3, after which the Acm group can be easily
removed using I2 and the second disulfide bond formed
between Cys 2 and 4. Note that the protection of the other
disulfide pair (i.e. Cys 1 and 3) initially with Acm works just as
well. More importantly, all these steps can be carried out in a
one-pot fashion, which significantly improved the overall yield.
With this strategy, we obtained all eight non-sY modified α-Ctx
in good yield and high purity. (Fig. S1, Table S2
)
Scheme
2. Regio-selective folding strategy using Acm
protecting group.
Figure 1. nP removal in peptide
Schematic presentation of the major species formed. (B)-(D):
1 using NH4OAc solution. (A)
Removal of nP from Cys-rich peptides by ammonium acetate
Analytical HPLC trace (214 nm) of the reaction after 0-21 h,
With the synthetic route developed for the non-modified α-
Ctx, we proceeded to the synthesis of the sY modified
peptides. Using AnIC (GGCCSHPACFASNPDYC) as an example,
respectively. Insert: MALDI-TOF-MS characterizations,
([M+H]+obs: 2018.9 Da, [M+H]+calc: 2021.6 Da); ([M+H]+
1
2
:
obs
1869.8 Da, [M-SO3+H]+calc: 1871.6 Da);
3
([M+H]+obs: 1868.1 Da,
we placed the Cys 4,17-Acm protected sY-peptide
literature conditions, i.e. 1-2 M NH4OAc solution, for nP
deprotection. Although the desired species was seen as the
major product in the solution (Fig. 1D) significant
1 under the
[M-SO3+H]+calc: 1869.6 Da) Note that the sulfate group will be
cleaved off under the laser used in MALDI. (E) Analytical HPLC
trace (214 nm) of the precipitation re-dissolved in 6 M Gnd-HCl
3
,
4
([M+H]+obs: 2017.1
precipitation, however, occurred soon after dissolving the
peptide (Fig. 1E). HPLC analysis indicates the presence of at
least 4 major species, 1-4 (Fig. 1). As mentioned earlier, we
reasoned that NH4OAc solution itself is not a probable buffer
and precipitation may occur depending on the property of the
solute peptides, which could also complicate the HPLC
and MALDI-TOF-MS characterization of
Da, [M+H]+calcd: 2019.6 Da). HPLC condition: 15%-40% MeCN
(with 0.1% TFA) in 25 min.
Removal of nP in aqueous solutions
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1889.8 Da. MS of
1739.8 Da. HPLC condition: 10%-40% MeCN (with 0.1% TFA) in
20 min.
6
: [M+H]+obs: 1739.8 Da, [M-SO3+H]+
:
calc
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We firstly tested the stability of the Fmoc-Tyr(OSO3-nP)-OH
monomer in MeCN/H2O system as well as a number of buffer
solutions (Fig. S7). It shows clearly that as the water content
increases, the extent of nP deprotection increases, with almost
complete removal when only H2O is used (Note that the
monomer is completely stable in MeCN, Fig. S7C). Further, a
number of buffer solutions (inclusive 20% MeCN for the
solubilization of the monomer), ranging from the routinely
used phosphate buffer, 6 M Gnd-HCl buffer and acetate buffer
to refolding buffer like NH4HCO3 buffer with oxidizing agents
were tested, and 55-70% of removal could be achieved within
24 hrs of the incubation. These results encouraged us to
proceed further with the deprotection of nP in a model
DOI: 10.1039/D0OB01526A
One-pot synthesis of sY modified α-Ctx
Having established that NH4HCO3 buffer is compatible with sY
deprotection, we employed a similar two-step oxidative
folding strategy as for the synthesis of the non-modified α-Ctx.
Taken the synthesis of sY-AnIC as an example, the nP
protected peptide
for nP removal as well as the formation of the disulfide bond
between Cys 1 and 3 under air oxidation. As shown in Fig. 3C
1 was incubated in 0.1 M NH4HCO3, pH 8.4
,
the nP group was completely removed after 4 hrs and the
disulfide bond was partially formed. When left longer, e.g. 24
hrs, the complete formation of disulfide bond could be
established (Fig. 3D). It is noted that for several α-Ctx peptides,
i.e. AnIA, AnIB and AuIA, the formation of the disulfide Cys1-
Cys3 was too slow and incomplete even after 48 hrs. In these
cases, the addition of oxidizing agent K3Fe(CN)6 (10 mM) was
found to be very effective in promoting the disulfide formation
peptide
5
containing an N-terminal sY modification
using selected buffers.
(sYDC(Acm)STNISPKQGLDK)
Gratifyingly, it was shown that under the conditions tested,
ranging from acidic to basic pHs, nP can be successfully
removed within 24 hrs, with very high conversion yields in H2O
and NH4HCO3 buffer (both over 90%, Fig. 2 C&F). It suggests
that nP removal might be achieved essentially in any aqueous
buffer, provided that the pH is probably controlled such that
the peptide itself is stable throughout the process.
(Fig. S8-S10). Care should be taken when adding K3Fe(CN)6,
cause excess of it might lead to peptide dimerization.
Next, the solution was acidified and the Acm deprotection
as well as the formation of the second disulfide bond was
promoted via the addition of I2. This process was usually very
efficient, e.g. in 5-10 min, and ascorbic acid was added to
quench the reaction. As shown in Fig. 3E, the I2 promoted Acm
deprotection and disulfide formation was very clean and the
transformation was almost quantitative. It is worth mentioning
that for peptides containing oxidation sensitive residues, i.e.
Met, the I2 treatment time should be kept minimum, which is
the case for sY-EpI (Fig. S11). Through this one-pot stepwise
disulfide pairing strategy, all eight sY α-Ctx containing peptides
were obtained with high purity and good overall yield (30-70%,
Fig. S8-S14, Table S3).
To our surprise, we did not find instability of the sulfate
ester on the modified α-Ctx peptides when the solution was
acidified (to pH 3) for the removal of Acm group, which
encouraged us to further purify the folded peptides using
normal MeCN/H2O (0.1% TFA) via semi-preparative HPLC (the
presence of sY modification was clearly indicated in the MS
traces under negative mode, Fig. 3C-E insert). While most
literature claim that sulfate ester is acid-labile,13,14,16,29,30 we
reason that it may be because of the tight tertiary structure of
α-Ctx, and with the help of H-bonding, that protects sY from
hydrolysis. Nevertheless, the present work suggests that the
sulfate ester group of the modified Tyr might not be acid-labile
in all the cases, which makes the purification process much
more convenient.
Figure 2 Analytical HPLC (214nm) and MALDI-TOF-MS of nP
removal from model peptide
(A). Schematic presentation of the nP removal. (B). Purifeid
model peptide (C). nP removal in H2O, pH 7.1. (D). nP
5 in different solutions for 24h.
5
.
removal in 0.1 M NaOAc and 0.1 M NaCl, pH 5.5. (E). nP
removal in 2 M NH4OAc, pH 7.0. (F). nP removal in 0.1 M
NH4HCO3, pH 8.4. MS of
5
: [M+H]+obs: 1889.1 Da, [M+H]+
:
calc
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not affect the serum stability, which may be crVuiecwiaAlrtifcolerOntlhinee
DOI: 10.1039/D0OB01526A
execution of its biological function under complex
physiological conditions.
Figure 4 Comparison of the serum stability between non-
modified α-Ctx and sY-containing α-Ctx.
Experimental
General methods
Amino acid, coupling reagent and resins for peptide synthesis
were obtained from GL Biochem. Fmoc-Tyr(OSO3nP)-OH was
synthesized by our own lab according to the literature (Fig. S2-
S6)16. General reagents were obtained from Sigma or Sangon,
HPLC grade MeCN was from Fisher. Horse serum was obtained
from Sangon. Analytical HPLC (Agilent 1260) was performed on
a Phenomenex Jupiter 4.6 μm C18 4.6×250 mm column
running at a flow rate of 1 mL/min with UV detection at 214
nm. The semi-preparative HPLC (Shimadzu AR-20) was
performed using a Phenomenex Jupiter 4.6 μm C18 10×250
mm column running at a flow rate of 5 mL/min with UV
detection at 214 nm. Both instruments were run using a
mobile phase composed of 0.1% TFA in H2O (Solvent A) and
0.1% TFA in MeCN (Solvent B) in a linear gradient as indicated.
The LC-MS was performed on Agilent LC/MSD (ESI) operating
in negative mode to detect the sulfate group. Low Resolution
MALDI-TOF mass spectra (Shimadzu 8020) were operated in
linear mode using a matrix of 10 mg/mL HCCA in water/MeCN
(1:1 v/v) with no TFA.
Figure 3 Synthesis of sY-AnIC. (A) Schematic presentation of nP
removal and regio-selective folding strategy. (B)-(D) Analytical
HPLC trace (214 nm) of the folding in NH4HCO3 after 0-24 hrs
incubation, respectively. Insert: ESI-MS characterization,
2H]2-obs: 1009.8 Da, [M-2H]2-calc: 1009.8 Da); ([M-2H]2-
974.8 Da, [M-2H]2-calc: 974.8 Da);
1 ([M-
2
:
obs
3
([M-2H]2-obs: 973.8 Da, [M-
2H]2-calc: 973.8 Da). (E) Analytical HPLC trace (214 nm)
following I2 treatment and ESI-MS characterization of sY AnIC
peptide ([M-2H]2-Obs: 901.75 Da, [M-2H]2-calc: 901.8 Da). HPLC
condition: 15%-50% MeCN (with 0.1% TFA) in 15 min.
Peptide Synthesis and folding
Peptides were synthesized by manual solid-phase Fmoc
chemistry on Rink Amide MBHA resins. Couplings were
performed using 4 eq of activated amino acid (HOBT: DIC:
amino acid, 1:2:1) relative to resin for about 2 hrs, While
Fmoc-Tyr(OSO3nP)-OH were performed using 2 eq of the
resins. In order to form two disulfides (Cys1-Cys3, Cys2-Cys4)
selectively, we used Trt group for the protection of Cys1 and
Cys3, and Acm group for Cys2 and Cys4. Fmoc deprotection
was carried out using 20% piperidine 2×5 min. Kaiser reagent
were used to test the extent of coupling, and typically a 0.5
Serum stability test of conotoxin
With all sY-containing α-Ctx in hand, we further tested and
compared the stability of the modified peptides with those
unmodified. The peptides were incubated together with 50%
serum solution at 37 C for 24 hrs, the resulting solution was
analyzed by HPLC for peptide integrity (Figs. S15-S22). As seen
from Fig. 4, similar to the non-modified peptides, at least 60%
of the sY-containing α-Ctx was intact after 24 hrs of the digest
by serum. It indicates that in all the cases sY modification does
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mmol/g loading was used. Following each coupling, capping or ester as instable under acidic conditions, the modified α-Ctx is
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deprotection step, the resin was washed with DMF×2, DCM×2, pretty robust when purified with eluentDsOcIo: n10t.a1i0n3i9n/gD00O.1B%015T2F6AA
DMF×2.
in our hands, and this will be crucial in obtaining the modified
Upon complete assembly of the peptide chain, the peptides peptides in large quantities. While previous reports have
were cleaved from the resin with TFA, DODT and H2O indicated the presence of sY modification in α-Ctx either via
(95:2.5:2.5, v/v/v) and incubated for 2h. At this point, the Acm LC-MS analysis or a possible sulfate electron density in the X-
groups of Cys and the nP group of Tyr were intact. Next, the ray crystallography structure,31 the investigation of its
resin was removed by filtration and volatile substances were biological consequence has rarely been possible. With the
removed by vacuum. The released peptides were precipitated strategy developed herein, these key questions can now be
and washed by cold ether, then centrifuged at 8,000 rpm for approached. While these studies are ongoing in our group, the
10 min. The precipitate was collected and lyophilized. The results will be reported should they become available.
obtained crude peptides were purified using RP-HPLC (This Moreover, the current strategy may find general applicability
step must be fast to prevent nP group from being removed in in the synthesis of other sY-containing peptides that also have
H2O/MeCN solution).
multiple Cys residues.
Afterwards, the linear peptides can be folded with correct
disulfide pairs by using a two-step oxidation strategy. To
remove nP group and form the first disulfide, we used a 0.1 M
NH4HCO3, pH 8.4 buffer and air-oxidation was applied here.
Peptides were dissolved to 1 mg/mL. While this condition was
enough for most α-Ctx, some α-Ctx peptides (AnIA, AnIB and
AuIA) couldn’t fold completely. In these cases, 10 mM
K3Fe(CN)6 (excessive amounts will cause peptide dimerization)
was added to promote the disulfide formation, and peptides
were dissolved to 0.3 mg/mL. In both of the above conditions,
the reaction was left for several hours and almost complete
conversion could be observed. Later, HCl was added the
solution to remove NH4HCO3 and pH adjusted to about 3.
Then, 0.05 eq of 0.1 M I2/MeOH was added to remove the Acm
group as well as to promote the second disulfide formation
and the solution immediately became cloudy. This reaction
takes just several minutes (The reaction time of EpI should be
kept as short as possible, because the Met contained in it can
be easily oxidized). Next, 1 M ascorbic acid was added until the
solution became clear. Finally, the folded peptides were
purified by semi-preparative HPLC.
Conflicts of interest
A Chinese patent is filed covering the synthetic method for sY
modified α-Ctx described in this manuscript (Application No.
CN201910944921.6).
Acknowledgements
This research was funded by the National Natural Science
Foundation of China (Nos. 81703406 and 91853117), the
Science and Technology Program of Guangzhou City (No.
201904010003) and partially by the National Key Research and
Development Program of China (No. 2017YFB002901).
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Organic & Biomolecular Chemistry
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DOI: 10.1039/D0OB01526A
Table of contents
Facile synthesis of the sulfotyrosine-containing -Conotoxins
Changpeng Lia, Chunmao Hea
aSchool of Chemistry and Chemical Engineering; South China University of Technology, Guangzhou 510640, China
A one-pot neopentyl deprotection and oxidative disulfide pairing strategy was developed for the facile synthesis of
sulfotyrosine (sY)-containing -Conotoxins (Ctx).