Revisiting ligation at selenomethionine: Insights into native chemical ligation at selenocysteine and homoselenocysteine

Article abstract of DOI:10.1016/j.bmc.2017.05.006

Selenomethionine (Sem) has been incorporated recombinantly into proteins many times to elucidate their structure and function. In this paper, we revisit incorporation via chemical protein synthesis to shed light on the mechanism of native chemical ligation. The effect of chalcogen position on ligation is investigated, and selenium-containing peptide ligation is optimized. Additionally, selective methylation is performed on selenolates in a peptide in the presence of unprotected thiols.

Full text of DOI:10.1016/j.bmc.2017.05.006

Accepted Manuscript
Revisiting ligation at selenomethionine: insights into native chemical ligation
at selenocysteine and homoselenocysteine
Rebecca Notis Dardashti, Norman Metanis
PII:
S0968-0896(17)30683-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.006
BMC 13724
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
30 March 2017
30 April 2017
4 May 2017
Please cite this article as: Dardashti, R.N., Metanis, N., Revisiting ligation at selenomethionine: insights into native
chemical ligation at selenocysteine and homoselenocysteine, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.05.006
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Revisiting ligation at selenomethionine:
insights into native chemical ligation at
selenocysteine and homoselenocysteine
Rebecca Notis Dardashti and Norman Metanis*
Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus Givat Ram, Jerusalem 91904, Israel

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Revisiting ligation at selenomethionine: insights into native chemical ligation at
selenocysteine and homoselenocysteine
Rebecca Notis Dardashtia and Norman Metanisa,*
aInstitute of Chemistry, The Hebrew University of Jerusalem, Safra Campus Givat Ram, Jerusalem 91904, Israel
E-mail: Metanis@mail.huji.ac.il
ARTICLE INFO
ABSTRACT
Article history:
Received
Selenomethionine (Sem) has been incorporated recombinantly into proteins many times to
elucidate their structure and function. In this paper, we revisit incorporation via chemical protein
synthesis to shed light on the mechanism of native chemical ligation. The effect of chalcogen
position on ligation is investigated, and selenium-containing peptide ligation is optimized.
Additionally, selective methylation is performed on selenolates in a peptide in the presence of
unprotected thiols.
Received in revised form
Accepted
Available online
Keywords:
Native chemical ligation
Selenomethionine
Selenocysteine
2009 Elsevier Ltd. All rights reserved
.
Chemical protein synthesis
Peptide methylation
with natural or unnatural moieties. For shorter peptides and
proteins, SPPS can be the sole method employed. For longer
sequences, however, ligation of unprotected protein segments
using methods such as native chemical ligation (NCL)8 can be
utilized.
1. Introduction
In protein chemistry, certain amino acids serve uniquely useful
roles in elucidating the structure, and hence the function, of the
proteins in which they reside. One such example is
selenomethionine (Sem), the selenium-containing analog of
methionine (Met). A product of plants’ processing selenium in
minerals, selenomethionine is an essential source of selenium for
many forms of life.1,2 Besides its nutritional value in nature,
selenomethionine has been used by scientists for years as a tool in
both NMR3 and crystallographic studies4 in order to uncover the
structure and function of proteins. In addition, a Sem and p-
cyanophenylalanine pair was recently used to probe protein
structure, wherein Sem quenches the fluorescence of p-
cyanophenylalanine via electron transfer, providing a sensitive
fluorescent probe to uncover helical structures in proteins.5 A
recent study6 utilizes a biocompatible, redox-based approach to
apply chemoselective modifications to Met; Se’s even lower
redox potential makes Sem an interesting candidate for future
applications.
NCL allows for the joining of two unprotected peptide
segments under mild conditions. In its proposed mechanism, the
sulfur of an N-terminal Cys on one peptide attacks the C-terminal,
labile thioester of the other segment. A subsequent, irreversible S-
to-N acyl shift yields a native peptide bond (Scheme 1). Cys’s
close relative, selenocysteine (Sec, U)9–11 is equally capable of
participating in NCL; this discovery has since been used in the
synthesis of natural and unnatural selenoproteins to shed light on
protein folding12,13 and other aspects of protein chemistry.14 As a
response to Sec and Cys’s relatively low occurrence in protein
sequences, a variation on NCL was developed in which the thiol
of Cys was removed following ligation to yield Ala.15,16 Similar
strategies were used to enable NCL using removable, thiol-
containing auxiliaries17–23 or the use of moieties with thiolated
side chains.24–34 More complex, multistep syntheses employing
thiazolidine35 or selenazolidine36 “masked precursors” have also
been employed to access difficult-to-synthesize proteins.
Sem is undoubtedly a useful tool for protein study, and it is
generally introduced into the protein sequence through
recombinant expression. However, such an approach replaces all
Met residues in the protein sequence with Sem. In addition, not
all proteins are easily expressed recombinantly. For these cases,
chemical protein synthesis (CPS) can be advisable. CPS, which
relies heavily on solid-phase peptide synthesis (SPPS),7 allows for
the building of proteins amino acid by amino acid, and easily
enables the chemist to swap out any amino acid in the sequence
In a similar vein, NCL was expanded to methionine.37,38 The
initial ligation was performed at homocysteine (hCys, hC) rather
than Cys, and subsequently methylated to give the native
methionine at the ligation site (Scheme 1). However, this method
was not useable on proteins containing Cys residues, as Cys’s
lower pKa meant it would surely undergo unwanted methylation
under the described conditions. In 2003, Roelfes and Hilvert

reported successful NCL at Sem using a homoselenocysteine
In order to investigate the effects of β- vs. γ-chalcogens on the
(hSec, hU) precursor,39 and since that time, to the best of our overall rate of NCL, a series of small peptides was designed.
knowledge, these findings were not utilized in CPS. However, we ZRAFS (Z = Cys, hCys, Sec, hSec) peptides were synthesized to
believe that with optimization, ligation at Sem can provide a allow analysis of the effect of the β- vs. γ-chalcogen position on
much-needed tool in contemporary protein study.
ligation as well as to see how thiol and selenol ligations differed
under our selected conditions. In addition, LYRAX-COSR (X =
Gly, Leu, Val) peptides were synthesized to see how rate of
reaction varied with steric hindrance of the thioester.43 In
accordance with standard protocol, all ligations were performed
with 3 mM peptides at pH slightly above 7 and in the presence of
thiol catalyst (250 mM MPAA).44 Normally, TCEP is also present
in reactions with Cys in order to avoid oxidation of the thiols and
encourage faster reactions. However, TCEP is known to remove
selenium in Sec,9,45,46 so TCEP is not normally used in Sec-
ligations. This reason, combined with Sec’s comparatively low
redox potential40 in comparison to Cys, and its tendency to form
stable diselenides under ambient conditions, mean that Sec
ligations proceed rather slowly in ambient conditions.9–11 Luckily,
in a recent paper,46 we reported that sodium ascorbate, a mild
radical quencher, successfully hinders the deselenization reaction.
For this reason, we included 50 mM TCEP and 100 mM sodium
ascorbate in all ligations.
Upon revisiting the paper, we chose to use it as a doorway to
further insight in CPS. Firstly, we noted that in ligations at Met
and Sem, the chalcogen attacking the thioester is in a γ, rather
than β, position on the amino acid side-chain. We wanted to
investigate further the effect this imposed on the ligation reaction,
and what light it shed on the proposed mechanism of NCL.
Secondly, under Roelfes and Hilvert’s reported conditions, the
ligation of hSec and a thioester was complete after five days.39 In
all likelihood, this can be attributed to the sensitivity of selenols
to air-oxidation and formation of stable diselenides.40 We
believed that this time could be improved using recent discoveries
made by our group. Thirdly, after noting the different pKa’s of
selenols and thiols,41,42 we wanted to show selective methylation
of hSec to Sem was possible in the presence of unprotected thiols.
This chemoselective methylation is not possible when
methylating hCys to Met, making the case for performing NCL at
Sem rather than Met even more powerful and useful.
Not surprisingly, ligations involving the sterically
unencumbered LYRAG-COSR proceeded the fastest, with all
reactions over 90% complete in under two hours (Figures 1a, S1-
S4, S17). No discernible difference in rate was apparent in any of
the Gly ligations; β- and γ-chalcogens appeared to ligate at similar
rates. Remarkably, both selenol and thiol containing peptides
ligated at identical rates, showing that the provided conditions can
successfully reduce ligation times at the previously sluggish Sec
and hSec.39
In this paper, we revisit ligation at selenomethionine to address
these points and better understand the nature of NCL.
Ligations performed at the bulkier LYRAL-COSR did take
longer – most ligations were complete between two and four
hours after beginning (Figures 1b, S5-S8, S18). While overall
rates did seem similar, we noted that ligation at hCys was only
70% complete at two hours when compared to all other ligations,
which were over 80% complete. This small but significant
difference cannot be explained by different energetics of a five-
vs. six-membered transition state of the reaction. Rather, the small
difference in rates here is more likely due to differences in pKa’s.
Cys is known to have a pKa of 8.3,41 whereas hCys has a slightly
higher pKa of 8.9.42 At ligation pH of 7, a larger percentage of
Cys (5.6%) compared to hCys (1.2%) will be deprotonated,
allowing it to perform the necessary first step of the reaction,
nucleophilic attack at the thioester, slightly faster. In this vein,
Sec and hSec ligations should be significantly faster due to their
low pKa’s.41 However, their lower redox potential,40 and resulting
sensitivity to oxidation and formation of diselenides, as explained
below, serves to slow their ligation rate significantly.
Our final set of ligations, performed at the quite sterically
hindered, β-branched Val in LYRAV-COSR, took significantly
longer (Figure 1c, S9-S12, S19).43 Both ligations involving thiols
were complete within 24 h, and, as a result, our selenol-
containing peptides were left to react for similar periods of time.
However, at 8 hours, some degree of deselenization was observed
at Sec and hSec ligations (URAFS and hURAFS, respectively),
and at 24 hours an even greater amount of deselenization was
seen. Taking this as an indication that some amount of sodium
ascorbate had decomposed over the longer course of the reaction,
we increased the concentration of this radical quencher to 200
mM, a fourfold excess over the reductant and deselenization
reagent, TCEP. We were pleased to note that this large excess of
sodium ascorbate was capable of inhibiting deselenization to a
satisfactory degree for 24 h. Still, selenol-containing peptides
Scheme 1. NCL at Cys, hCys, Sec, and hSec. Methylation after NCL
at hCys and hSec will provide Met and Sem.
2. Results and Discussion

were significantly slower in ligating when compared to thiol- bond to a selenolate – according to the pKa of Sec, 5.2,41 the vast
containing peptides, with the selenol-containing peptides being majority of selenols will be deprotonated at pH 7. The selenolate
must immediately perform a nucleophilic attack on a thioester in
solution to avoid being immediately re-oxidized by ambient
oxygen present in the system. The rapid rate of re-oxidation is a
problem only for selenolate; thiols have a much higher redox
potential and are not as rapidly oxidized.40 In relatively
unhindered thioesters, such as those adjacent to Gly or Leu, the
nucleophilic attack occurs rapidly enough that there is no
observed difference between S and Se ligations. However, the
bulky Val causes the nucleophilic attack to proceed much more
slowly. Thus, the overall rate of Se ligations to Val-adjacent
thioesters is doubly slow – hindered by both the difficulty for Sec
or hSec to attack at the bulky, β-branched amino acid and the
subsequent re-oxidation of Sec or hSec to a diselenide, which
must be reduced again by TCEP to re-initiate ligation.
On the whole, a difference between the rate of ligation of
chalcogens at either β- or γ-positions was observed in the two sets
of longer ligations (at Leu and Val), but the difference was small
and hence more easily attributed to a difference in pKa, rather than
in the kinetics of transition state. Nevertheless, we were able to
reduce the ligation time of selenol-containing peptides to be
similar to that of thiol-containing peptides. In addition, we found
that the radical quencher sodium ascorbate has a tendency to
degrade over time in traditional ligation conditions, and must be
present in high concentrations to prevent deselenization in the
presence of TCEP for overnight reactions.
In order to test the selectivity of selenol methylation in the
presence of thiols – a property never investigated in previous
works – a model peptide with sequence Ala-His-hSec-Ser-Tyr-
Lys-Trp-Cys-Asp-Met-Ala-NH2, which contains all potentially
sensitive residues, was synthesized and subjected to various
methylation conditions. All methylation studies were performed
in 0.2 M phosphate buffer with 6 M guanidinium hydrochloride,
50 mM TCEP, and 300 mM sodium ascorbate. We first
investigated the effect of providing different concentrations of the
methylating agent, 4-methyl nitrobenzene sulfonate (MNBS) at
pH 5 (Figures S20-S22). Reactions were performed in 1, 10, and
100 mM MNBS (to 1 mM peptide). As expected, the 1 mM
reaction was the slowest and the 100 mM was fastest. However,
unwanted side-reactions such as deselenization and Sem
oxidation were observed at the highest concentration of MNBS.
As a result, we chose to use 10 mM MNBS – the middle ground –
in all reactions going forward.
We next studied the effect of temperature on methylation;
reactions were performed at 25, 37, and 50 °C at pH 6 (Figures
S25-27). Deselenization, due to the presence of TCEP in solution,
was observed in all reactions. Reactions performed at 37 and 50
°C showed levels of deselenization that were unacceptable (20%
deselenized at 4 h at 37 °C, and 35% deselenized at 4 h at 50 °C),
while the reaction at 25 °C was more stable (8% deselenized at 4
h), although slower.
Further, we investigated selectivity of the methylation reaction
Figure 1. Comparison of Cys, hCys, Sec, and hSec ligations at Gly,
as a function of pH. As methylation proceeded quite slowly at pH
5, we chose to investigate the methylation reaction at pH 6, 7, and
8 with 10 equiv MNBS and at 25 °C (Figures S23-S25). At pH 6,
the peptide was approximately 5% dimethylated at 2 h; this
number did not change when the reaction was checked at 4 h and
8 h. In comparison, the reaction at pH 7 showed 8%
dimethylation at 8 h and 43% dimethylation at pH 8 at 8 h (Figure
2). In accordance with previous studies,45 deselenization was also
observed to increase with pH. While the peptide was only 6%
deselenized at pH 6 after 8 h, it was 8% deselenized at pH 7 and
38% deselenized at pH 8 at the same time point. During
Leu, and Val thioesters. In (a-c) the s.d. values were calculated from
experiments done in triplicate and the lines connecting the data
points are shown only for clarity and do not represent a data fit.
Comparison of ZRAFS peptides over time can be seen in in Fig. S13-
S16.
only 30% consumed at 8 hours of reaction and the thiol-
containing peptides, over 60% (Figure 1c).
This phenomenon may be explained when the entire system of
the ligation reactions is considered. TCEP reduces the diselenide

screenings for MNBS equivalents at pH 5, dimethylation that the number of atoms in the ring transition state of native
(methylation of the selenol and the thiol) was not detected. chemical ligation has no observable impact on the overall rate of
Selectivity of the methylation reaction was confirmed via trypsin reaction. This supports the proposed mechanism, in which the
digest [Figure S28].
rate-determining step of NCL is the attack of the thioester by thiol
or selenol. Indeed, we were able to observe the impact of steric
hindrance in the significantly longer ligation times at Val when
compared to ligations at Leu and Gly. The impact of Val’s
bulkiness was compounded when ligations with the easily
oxidized Sec or hSec were performed – these ligations took
significantly longer than ligations at Val thioester with Cys or
hCys. For less bulky amino acids, we successfully found
conditions that reduced the ligation time of the easily-oxidized
selenol to equal that of thiols.
Additionally, by exploiting unique properties of selenols –
most importantly their lower pKa values – we were able to
determine which conditions allowed for selective methylation of
hSec in the presence of unprotected Cys as well as other reactive
amino acid side chains. We applied optimized ligation and
methylation conditions in the synthesis of the NEDD8 protein,
with satisfactory yields.
4. Experimental
Figure 2. Selectivity of selenol methylation in the model peptide Ala-
His-hSec-Ser-Tyr-Lys-Trp-Cys-Asp-Met-Ala-NH2. Comparisons of
chromatograms show the dramatic effect of pH on hSec methylation
in the model peptide.
1.1. Homoselenocysteine Synthesis
Boc- and Fmoc-hSec(Mob)-OH were synthesized from
selenomethionine following previously published procedures.39,49
1. 1.1. Homoselenocystine
It was thus determined that pH, rather than temperature or
equivalents of methylating reagent, has the most influence over
3.06 g selenomethionine (15.6 mmol) was stirred in 150 mL
the rate of methylation of hSec to Sem. This is easily explained liquid ammonia at -78 oC. Small pieces of 0.9 g sodium metal (39
mmol, 2.5 equiv) were added slowly over the course of 20
minutes, and the reaction was stirred for 1 h. 3.8 g solid NH4Cl
by the different pKa’s of the amino acids. As mentioned above,
Cys’s pKa of 8.341 and hCys’s pKa of 8.942 mean that the majority
of both thiols are protonated, thus not susceptible to methylation, was added to neutralize any NaNH2 in solution. The reaction was
at pH’s 6, 7, or 8 investigated here. The significantly lower pKa’s stirred in the fume hood at room temperature overnight to
of Sec, 5.2,41 and hSec, which has not been formally determined evaporate any remaining NH3, leaving a yellow solid. 80 mL H2O
but likely is 3 units lower than hCys (~6), mean that deprotonated was added and stirred to form a suspension. N2 gas was bubbled
through and passed through a wash bottle filled with bleach to
remove any Me2Se. pH was lowered to 1 with concentrated HCl,
then 40 mL H2O was removed via rotary evaporation. Solution
was adjusted to pH 6 with 2 N NaOH and stirred for 30 minutes.
The resulting yellow precipitate was filtered and lyophilized to
dryness. 1.77 g of light yellow solid homoselenocystine was
retrieved (62% yield).
selenolates undergo methylation at pH’s in which thiols are still
protonated and thereby unreactive.
With the optimized conditions for hSec ligation and
methylation in hand, we then chose to apply our findings in the
synthesis of the post-translationally modifying protein, NEDD8.
NEDD8 is a relatively small ubiquitin-like protein (76 amino
acids) with a Met at position 50, which we chose to substitute
with Sem. NEDD8 was prepared from two peptide segments:
NEDD8(2-49)-COSR, which was prepared using an N-acylurea
precursor to a thioester47,48 and NEDD8(50-76)(Met50hSec).
NEDD8(2-49)-COSR was prepared in 3% yield, and NEDD8(50-
76)(Met50hSec) was prepared in similar yield, 4%, which was
attributed to the poor coupling of Fmoc-hSec(Mob)-OH. Ligation
was performed under conditions similar to those used on the short
peptides – 3 mM of each peptide segment, 50 mM TCEP, and 100
mM sodium ascorbate in a 0.2 M phosphate buffer containing 6
M guanidinium hydrochloride and 250 mM MPAA. We realized
that the ligation conditions were similar to those applied in our
methylation experiments, and we were pleased to note that,
following ligation, hSec50 could be methylated in a one-pot
manner for a total yield of 28%. In comparison, purification of
ligated product and subsequent methylation resulted in an overall
yield of 26%.
1. 1.2. Se-p-methoxybenzyl-homoselenocysteine
hydrochloride
Under Ar atmosphere, 0.5 g of homoselenocystine (1.39
mmol) was suspended in 2 mL 0.5 N NaOH and cooled to 0 oC in
an ice bath. 0.45 g NaBH4 (11.7 mmol, 8.4 equiv) in 4 mL H2O
was added dropwise over 15 minutes to the cooled, stirring
solution. The solution was adjusted to pH 6 with glacial acetic
acid, then 371 µL 4-methoxybenzyl chloride (2.71 mmol, 1.95
equiv) was added in one aliquot. The solution was stirred for 1.5 h
o
at 0 C. The solution was adjusted to pH 1-2 with concentrated
HCl. The white precipitate was filtered and washed with a small
amount of H2O, then lyophilized to dryness. 414 mg of white
solid was obtained (quantitative yield) and taken to the next step
without further purification.
1. 1.3. Nα -tBoc-Se-p-methoxybenzyl-
homoselenocysteine
290 mg NH2-hSec(Mob)-OHꢀHCl (0.86 mmol) was dissolved
in a solution of 224 mg NaHCO3 (2.66 mmol, 3 equiv) in 5.8 mL
H2O at 0 oC. A solution of 307 µL Boc-anhydride (1.33 mmol, 1.5
equiv) in 5.8 mL dioxane was added dropwise to the stirring
solution over the course of 1 hour. The solution was allowed to
3. Conclusions
In this work, we used the little-studied ligation at
selenomethionine as a stepping-stone into understanding more
about the reactions connected to it. We were able to determine

warm to room temperature and stir overnight. The reaction was 1. 2.2. Peptide Thioester Synthesis
extracted once with 12 mL ether. The ether layer was washed
once with saturated aqueous NaHCO3. The aqueous layers were
combined and stirred over ice. 1 N HCl was added slowly, due to
CO2 formation, until pH 1. Product was extracted twice in ethyl
acetate, which was dried over MgSO4 and evaporated. The
resulting yellowish oil was redissolved in a combination of H2O
and ACN, then lyophilized to give 202 mg yellow powder (0.503
mmol, 59% yield) of Boc-Sec(Mob)-OH.
Peptide thioesters were synthesized according to the
procedures outlined in the literature from an N-acylurea
precursor.47,48 Mono-Fmoc-(3,4)-diaminobenzoic acid (Fmoc-
Dbz-OH) or Fmoc-3-amino-4-(methylamino)benzoic acid (Fmoc-
MeDbz-OH) (4 equiv) was activated with HATU (4 equiv) and
DIEA (8 equiv) in DMF for 3 minutes, then coupled to the
deprotected resin for 2 hours. Following peptide synthesis, the
peptidyl-resin was washed once in DMF, then twice in DCM. A
solution of p-nitrophenyl chloroformate (5 equiv) in DCM (4
mL/0.1 mmol) was added, shaken for 1 hour, and then drained
and washed well with DCM. This step was repeated two more
times, after which a solution of 0.5 M DIEA in DMF (2 mL/0.1
mmol) was added and shaken for 30 min to complete the
cyclization process. The peptide was cleaved from resin and
converted to a thioester without undergoing any additional
purification.
1.1.4. Nα -(9H-Fluoren-9-ylmethoxycarbonyl)-Se-p-
methoxybenzyl homoselenocysteine
418 mg (1.23 mmol) NH2-hSec(Mob)-OHꢀHCl salt was
dissolved in 5 mL of 10% m/v NaHCO3 in H2O and cooled to 0
oC. A solution of 415 mg (1.23 mmol, 1 equiv) Fmoc-ONSu was
dissolved in 3.5 mL dioxane and added dropwise to the cold,
stirring solution. The reaction was warmed to room temperature
and stirred overnight, forming a white solid. The mixture was
extracted twice with ether, and the aqueous layer was adjusted to
pH 1-2 with concentrated HCl. This was extracted three times
with ethyl acetate. The ethyl acetate layers were combined and
washed twice with 1 N HCl and twice with H2O, then dried over
MgSO4. The solvent was evaporated and the yellow oil was
resuspended in H2O and ACN, then lyophilized to give 391 mg
beige powder (61% yield) of Fmoc-Sec(Mob)-OH, which was
characterized by 1H- and 13C-NMR [Figures S34-35].
Crude peptide-Nbz was dissolved in phosphate buffer (0.2 M,
6 M GdmCl, pH ~ 7) at 3-10 mM concentration. Methyl ester of
3-mercaptopropionic acid (MMP) was added (5% v/v) and the
solution was incubated at room temperature for 1-48 h and
monitored via HPLC. Once reaction was deemed to be complete,
the peptide-thioester was purified via RP-HPLC.
1. 2.3. Synthesis of LYRAX Peptides
1. 2.3.1. LYRAG-MMP
1.2. Peptide Syntheses
LYRAG-MMP was synthesized from an Me-Dbz precursor on
a 0.1 mmol scale. Following peptide synthesis, Nbz formation
was performed as described above to give LYRAG-MeNbz-resin.
The resin was cleaved according to general procedure and
afforded crude LYRAG-MeNbz (65 mg). The cleaved peptide
was treated with MMP according to the described procedure to
give the corresponding LYRAG-MMP. This peptide was purified
by prep HPLC (15%-25% B over 55 min). 32.3 mg (47% yield)
pure peptide was obtained, and its purity was verified by
analytical HPLC.
1.2.1. General Peptide Synthesis
All peptides were synthesized according to standard Fmoc-
SPPS procedure, manually or on an automated peptide
synthesizer. Unless otherwise indicated, Tentagel Rink Amide
Resin was used and syntheses were performed on a 0.25 mmol
scale. Standard deprotections were performed twice in 20%
piperidine in DMF for five minutes each time. For manual
synthesis, Fmoc-amino acids (1 mmol, 4 equiv) were activated for
3 minutes with HATU or HCTU (1 mmol, 4 equiv in 2.5 mL
DMF) and DIEA (2 mmol, 8 equiv in 2.5 mL DMF), then shaken
to couple for 30 minutes. For automated synthesis, Fmoc-amino
acids (2 mmol, 8 equiv) were activated for 3 min with HATU or
HCTU (2 mmol, 8 equiv in 5 mL DMF) and DIEA (4 mmol, 16
equiv in 5 mL DMF), then shaken to couple for 30 minutes.
Amino acids containing selenium (0.375 mmol, 1.5 equiv) and
OxymaPure (0.375 mmol, 1.5 equiv) were dissolved in 5 mL 1:1
DMF:DCM, cooled to 0oC, and then activated with DIC (0.35
mmol, 1.4 equiv) for 5 minutes and shaken to couple for 2-4
hours.
1. 2.3.2. LYRAL-MMP
LYRAL-MMP was synthesized from a Dbz precursor on a
0.25 mmol scale. Following peptide synthesis, Nbz formation was
performed as described above to give LYRAL-Nbz-resin. The
resin was cleaved according to general procedure and afforded
crude LYRAL-Nbz (164 mg). 55.5 mg of cleaved peptide was
treated with MMP according to the described procedure to give
the corresponding LYRAL-MMP. This peptide was purified by
prep HPLC (15%-40% B over 55 min). 23.7 mg (38% yield) pure
peptide was obtained, and its purity was verified by analytical
HPLC.
For cleavage of 200 mg resin, 13 mL cleavage cocktail was
prepared (95% TFA, 2.5% H2O, 2.5% TIPS). If Cys or hCys was
present in the sequence, a cocktail consisting of 94% TFA, 2.5%
H2O, 1% TIPS, and 2.5% EDT was used. Cocktail was added to
peptidyl-resin and shaken for 3 h. The resin was removed by
filtration, and N2 gas was bubbled through to remove TFA,
followed by addition of cold ether to precipitate peptide. Mixture
1. 2.3.3. LYRAV-MMP
LYRAV-MMP was synthesized from a Dbz precursor on a
0.25 mmol scale. Following peptide synthesis, Nbz formation was
performed as described above to give LYRAV-Nbz-resin. The
resin was cleaved according to general procedure and afforded
crude LYRAV-Nbz (216.4 mg). 54.6 mg cleaved peptide was
treated with MMP according to the described procedure to give
the corresponding LYRAV-MMP. This peptide was purified by
prep HPLC (15%-40% B over 55 min). 22.7 mg (50% yield) pure
peptide was obtained, and its purity was verified by analytical
HPLC.
o
was shaken to create one phase, then chilled at -20 C for 30
minutes before centrifugation at 5000 RPM for 10 minutes. After
decanting ether, peptide was resuspended in 50% H2O and 50%
ACN and lyophilized to dryness. At this point, peptides
containing selenium underwent a second cleavage in neat TFA
and 2 equiv DTNP for 3 h,50 followed by similar treatment
described above to give dry, lyophilized product. The resulting
crude peptide was dissolved in either aqueous ACN or phosphate
buffer containing guanidinium hydrochloride and purified via
preparative RP-HPLC.
1. 2.4. Synthesis of ZRAFS Peptides

1.2.4. 1. CRAFS-NH2
with 250 mM MPAA. The buffer was adjusted to pH 7.1 prior to
addition of peptides. 10 uL aliquots of reaction mixture were
removed at predetermined time points and quenched in 50-70 uL
of 1% TFA in a 1:1 H2O:ACN solution before HPLC analysis.
The yields are calculated based upon the integration of the HPLC-
traces under consideration of the extinction coefficients. (ε280
(peptide thioester) = ε280 (cysteine peptide) = 1280 L·mol-1·cm-1;
ε280 (ligation product) = 2560 L·mol-1·cm-1).51
CRAFS-NH2 was synthesized from TentaGel R RAM Resin
on a 0.1 mmol scale and cleaved according to general procedure
to give 74.3 mg crude peptide, which was purified by prep HPLC
(8%-33% B over 55 min). 54 mg pure peptide was obtained (93%
yield), and its purity was verified by analytical HPLC.
1.2.4. 2. hCRAFS-NH2
hCRAFS-NH2 was synthesized from TentaGel R RAM Resin
on a 0.1 mmol scale according to general procedure. 1.5 equiv
Fmoc-hCys(Trt)-OH was activated with 1.5 equiv HATU and 3
equiv DIEA in DMF for 3 min, then shaken with peptidyl-resin
for 1.5 h to couple. Peptidyl-resin was cleaved according to
1.4. Methylation of AlkPep
1. 4.1. Optimized Procedure
Methylations were performed at 1 mM concentration of
general procedure to give 74.3 mg crude peptide, which was peptide with 10 mM methyl nitrobenzene sulfonate (MNBS) in an
purified by prep HPLC (8%-33% B over 55 min). 45 mg pure aqueous buffer containing 6 M GdmCl and 0.2 M phosphate
peptide was obtained (75% yield), and its purity was verified by buffer with 10% acetonitrile and at 25 oC. 50 mM TCEP and 300
analytical HPLC.
mM NaAsc were dissolved in the buffer and pH was adjusted to 6
prior to addition of peptide.
1.2.4. 3. URAFS-NH2
1. 4.2. Equivalents MNBS Screen
URAFS-NH2 was synthesized from TentaGel R RAM Resin
on a 0.1 mmol scale according to general procedure. 1.5 equiv
Boc-Sec(Mob)-OH was activated with 1.5 equiv OxymaPure and
Methylations were performed at 1 mM concentration of
peptide at room temperature as described above with 1, 10, or 100
mM MNBS in an aqueous buffer containing 6 M GdmCl and 0.2
M phosphate buffer with 10% acetonitrile. 50 mM TCEP and 300
mM NaAsc were dissolved in the buffer and pH was adjusted to 5
prior to addition of peptide.
o
1.4 equiv DIC in 1:1 DCM:DMF for 5 minutes at 0 C, then
shaken with peptidyl-resin for 2 hours to couple. Peptidyl-resin
was cleaved according to general procedure to give 102.7 mg
crude peptide. This peptide was dissolved in aqueous acetonitrile
and treated with 1:2 NaAsc:TCEP prior to purification in 1. 4.3. Temperature Screen
preparative RP-HPLC (3%-28% B over 55 min). 34 mg pure
peptide (54% yield) was obtained, and its purity was verified by
analytical HPLC.
Methylations were performed at 1 mM concentration of
peptide with 10 mM MNBS in an aqueous buffer at pH 6 as
described above at three different temperatures: 25o, 37o, and
50oC.
1.2.4. 4. hURAFS-NH2
1. 4.4. pH Screen
hURAFS-NH2 was synthesized from TentaGel R RAM Resin
on a 0.1 mmol scale according to general procedure. 1.2 equiv
Boc-hSec(Mob)-OH was activated with 1.2 equiv OxymaPure
Methylations were performed at 1 mM concentration of
peptide with 10 mM MNBS at 25oC in an aqueous buffer as
described above with the pH adjusted to 6, 7, or 8 prior to
addition of peptide.
o
and 1.1 equiv DIC in 1:1 DCM:DMF for 5 minutes at 0 C, than
shaken with peptidyl-resin for 2 hours to couple. Peptidyl-resin
was cleaved according to general procedure to give 101.3 mg
crude peptide. This peptide was dissolved in aqueous acetonitrile
and treated with 1:2 NaAsc:TCEP prior to purification in
preparative RP-HPLC (9.5%-34.5% B over 55 min). 35 mg pure
peptide (54% yield) was obtained, and its purity was verified by
analytical HPLC.
1.5. Synthesis of NEDD8(Met50Sem) Protein
NEDD8(Met50Sem) was synthesized from two segments:
NEDD8(2-49)-COSR and NEDD8(50-76)(Met50hSec). The two
were ligated and the product was methylated to give the final
NEDD8(Met50Sem).
1. 5.1. NEDD8(2-49)-COSR
1.2.5. Synthesis of AlkPep
NEDD8(2-49)-COSR was synthesized on a 0.125 mmol scale
on an automated peptide synthesizer using N-acylurea precursor
Dbz on TentaGel R RAM Resin, converted to Nbz, and cleaved
from resin as described above. 410 mg of crude NEDD8(2-49)-
Nbz was obtained. The dry peptide was dissolved in 15.2 mL
buffer (6 M GdmCl, 0.2 M phosphate buffer) and 800 µL MMP
was added. Reaction was observed to be complete after 5 h, and
purified using preparative RP-HPLC, in 20%-45% B over 55
minutes (Figure S29). 20.7 mg pure NEDD8(2-49)-MMP (3%
yield) was obtained.
AlkPep
(Ala-His-hSec-Ser-Tyr-Lys-Trp-Cys-Asp-Met-Ala-
NH2) was synthesized from TentaGel R RAM Resin on a 0.1
mmol scale according to general procedure. 1.5 equiv Fmoc-
hSec(Mob)-OH was activated with 1.5 equiv OxymaPure and 1.4
o
equiv DIC in 1:1 DCM:DMF for 5 minutes at 0 C, then shaken
with peptidyl-resin for 2 hours to couple. Peptidyl-resin was
cleaved according to general procedure to give 114.4 mg crude
peptide. This peptide was dissolved in aqueous acetonitrile and
treated with 1:2 NaAsc:TCEP prior to purification in preparative
RP-HPLC (9.5%-34.5% B over 55 min). 20.6 mg pure peptide
(15% yield) was obtained, and its purity was verified by
analytical HPLC.
1. 5.2. NEDD8(50-76)(Met50hSec)
NEDD8(50-76)(Met50hSec) was synthesized on Fmoc-Gly
Wang Resin at a 0.25 mmol scale on an automated peptide
synthesizer according to procedures described above. The final
amino acid, Fmoc-hSec(Mob)-OH, was coupled using
OxymaPure and DIC as described. Cleavage was performed in
two steps. First, peptidyl-resin was shaken for 2 h in 14 mL
cleavage cocktail (2.5% TIPS, 2.5% H2O, 95%TFA). Peptide was
1.3. Ligations of Peptides – general procedure
Ligations were performed at 3 mM concentration of each
peptide in 6 M GdmCl and 0.2 M phosphate buffer, with the
addition of 50 mM TCEP and 200 mM sodium ascorbate (NaAsc)

Nichiporuk, R. V.; Iavarone, A. T.; Wells, J. A.; Toste, F. D.; Chang, C.
dried and then shaken for 3 hours in TFA with 2 equiv DTNP.
200 mg of the 600 mg crude peptide was purified via prep RP-
HPLC (22%-57% B over 55 minutes) giving 30 mg pure peptide
(4% yield) (Figure S30).
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1.5.3. Ligation
10 mg of NEDD8(2-49)-MMP and 5 mg of NEDD8(50-
76)(Met50hSec) were dissolved in 577 µL (to about 3 mM of
each peptide) of a buffer containing 6 M GdmCl, 0.2 M
phosphate buffer, 50 mM TCEP, 100 mM NaAsc, and 250 mM
MPAA. The pH was adjusted to 7.1 and the reaction was allowed
to proceed overnight.
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1.5.4. One-pot ligation and methylation
Following overnight ligation, 100 µL of the reaction mixture
was taken and 90 µL of fresh buffer containing 6 M GdmCl, 0.2
M phosphate buffer, 50 mM TCEP, and 100 mM NaAsc were
added. 12.5 µL 1 M MNBS in ACN was added to the mixture to
give a final solution of 1.5 mM peptide, 75 mM MNBS, and 7.5%
ACN in aqueous buffer. After 22 h, methylation was still not
complete. An additional 12.5 µL 1 M MNBS was added and
reaction was completed overnight (Figure S33) and purified via
semi-prep RP-HPLC (Figure S32). 0.7 mg final product was
obtained, for an overall yield of ligation and methylation of 28%.
Metanis, N.; Hilvert, D. Angew. Chemie - Int. Ed. 2012, 51 (23),
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1.5.5. Two-step methylation and ligation
NEDD8(2-76)(Met50hSec) was purified via semi-prep RP-
HPLC (Figure S31). This was methylated according to the
optimized procedure described above and again purified via RP-
HPLC (Figure S32). In total, 3.2 mg of material was recovered,
for an overall yield of ligation and methylation of 26%.
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Acknowledgements
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This work was supported by Israel Science Foundation
(1072/14), the US-Israel Binational Science Foundation (BSF)
(2014167), and the German-Israeli Foundation for Scientific
Research and Development (GIF) (I-1355-302.5/2016). R.N.D.
thanks the Kaete Klausner Fellowship for financial support. We
thank Orit Ktorza and Reem Mousa for their support, Israel
Alshanski for NMR assistance, and Dr. Post Sai Reddy for
assistance in homoselenocystine synthesis.
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