2,2'-Dithiobis(5-nitropyridine) (DTNP) as an effective and gentle deprotectant for common cysteine protecting groups

Article abstract of DOI:10.1002/psc.1403

Of all the commercially available amino acid derivatives for solid phase peptide synthesis, none has a greater abundance of side-chain protection diversity than cysteine. The high reactivity of the cysteine thiol necessitates its attenuation during peptide construction. Moreover, the propensity of cysteine residues within a peptide or protein sequence to form disulfide connectivity allows the opportunity for the peptide chemist to install these disulfides iteratively as a post-synthetic manipulation through the judicious placement of orthogonal pairs of cysteine S-protection within the peptide's architecture. It is important to continuously discover new vectors of deprotection for these different blocking protocols in order to achieve the highest degree of orthogonality between the removal of one species in the presence of another. We report here a complete investigation of the scope and limitations of the deprotective potential of 2,2'-dithiobis(5-nitropyridine) (DTNP) on a selection of commercially available Cys S-protecting groups. The gentle conditions of DTNP in a TFA solvent system show a remarkable ability to deprotect some cysteine blocking functionality traditionally removable only by more harsh or forcing conditions. Beyond illustrating the deprotective ability of this reagent cocktail within a cysteine-containing peptide sequence, the utility of this method was further demonstrated through iterative disulfide formation in oxytocin and apamin test peptides. It is shown that this methodology has high potential as a stand-alone cysteine deprotection technique or in further manipulation of disulfide architecture within a more complex cysteine-containing peptide template.

Full text of DOI:10.1002/psc.1403

Research Article
Received: 21 March 2011
Revised: 21 June 2011
Accepted: 29 June 2011
Published online in Wiley Online Library: 14 November 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1403
2,2′-Dithiobis(5-nitropyridine) (DTNP) as
an effective and gentle deprotectant for
common cysteine protecting groups
Alayne L. Schroll,a Robert J. Hondalb,c* and Stevenson Flemer Jr.a,c*
Of all the commercially available amino acid derivatives for solid phase peptide synthesis, none has a greater abundance of
side-chain protection diversity than cysteine. The high reactivity of the cysteine thiol necessitates its attenuation during pep-
tide construction. Moreover, the propensity of cysteine residues within a peptide or protein sequence to form disulfide con-
nectivity allows the opportunity for the peptide chemist to install these disulfides iteratively as a post-synthetic manipulation
through the judicious placement of orthogonal pairs of cysteine S-protection within the peptide’s architecture. It is important
to continuously discover new vectors of deprotection for these different blocking protocols in order to achieve the highest
degree of orthogonality between the removal of one species in the presence of another. We report here a complete investi-
gation of the scope and limitations of the deprotective potential of 2,2′-dithiobis(5-nitropyridine) (DTNP) on a selection of
commercially available Cys S-protecting groups. The gentle conditions of DTNP in a TFA solvent system show a remarkable
ability to deprotect some cysteine blocking functionality traditionally removable only by more harsh or forcing conditions.
Beyond illustrating the deprotective ability of this reagent cocktail within a cysteine-containing peptide sequence, the utility
of this method was further demonstrated through iterative disulfide formation in oxytocin and apamin test peptides. It is
shown that this methodology has high potential as a stand-alone cysteine deprotection technique or in further manipulation
of disulfide architecture within a more complex cysteine-containing peptide template. Copyright © 2011 European Peptide
Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: cysteine; protecting group; deprotection; DTNP; thioanisole; thiol
that each pair of cysteine thiol protecting groups are orthogonal
to each other so that one motif can be removed under conditions
Introduction
to which the remaining blocking protocol are robust. As such, it is
desirable to either expand the number of available cysteine pro-
tection protocols or to devise more attractive deprotection condi-
tions for existing protection schemes in order to allow the
highest degree of blocking-group diversity.
We previously reported a new, gentle methodology for the re-
moval of acetamidomethyl (Acm) and p-methoxybenzyl (Mob)
cysteine protection that did not require forcing removal condi-
tions standard for these protecting groups [6]. In this approach,
treatment of Cys(Mob)- or Cys(Acm)-containing peptides with
varying concentrations of 2,2′-dithiobis(5-nitropyridine) (DTNP)
Cysteine (Cys) is a common amino acid, possessing an elevated
level of importance in the general function of peptides and pro-
teins because of its ability to form disulfide connectivity with other
cysteine residues within the protein’s primary sequence. Specific
disulfide architecture is crucial to the effective functioning of many
essential proteins as well as for various peptide hormones and
other messenger molecules. In order to gain a greater understand-
ing of disulfide-containing peptide systems, it is frequently neces-
sary to carry out their linear construction through solid phase pep-
tide synthesis (SPPS) with proper disulfide closure being brought
about as a post-synthetic manipulation. Although there are many
proven methodologies for installing post-synthetic disulfide archi-
tecture into peptides [1–3], the effectiveness of each approach
can be heavily dependent upon the size and sequence of the
peptide chain. When multiple disulfide connectivity must be
implemented correctly in a stepwise fashion, the avenue through
which this is carried out can be anything but trivial.
For no amino acid side-chain is there a greater abundance of
side-chain protecting protocol as there is for cysteine [4,5]. This
is in part because of the multiple levels of protecting group or-
thogonality necessary for the requisite construction of multiple
disulfide-containing peptides. In a stepwise approach toward en-
try into multiple disulfide constructs, it often becomes necessary
to employ orthogonally protected cysteine pairs to effect one
disulfide closure at a time. For a peptide target in which it is de-
sirable to install each disulfide bond sequentially, it is important
*
Correspondence to: Stevenson Flemer Jr., Department of Biochemistry, Room
B413 Given Bldg, 89 Beaumont Ave, University of Vermont, Burlington, VT
05405, USA. E-mail: Robert.Hondal@uvm.edu
** Robert J. Hondal, Department of Biochemistry, University of Vermont, College
of Medicine, 89 Beaumont Ave, Room B415, Given Bldg, Burlington, VT
05405, USA. E-mail: Robert.Hondal@uvm.edu
a Department of Chemistry, One Winooski Park, Saint Michael’s College,
Colchester VT 05439, USA
b Department of Biochemistry, University of Vermont, Room B415 Given Bldg, 89
Beaumont Ave Burlington VT 05405, USA
c
Department of Biochemistry, University of Vermont, Burlington, Room B415
Given Bldg, 89 Beaumont Ave VT 05405, USA
J. Pept. Sci. 2012; 18: 1–9
Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

SCHROLL, HONDAL AND FLEMER
[7] in a 2% thioanisole/TFA solvent resulted in virtually complete
deprotection within 1 h. In this work, we carried out a methodical
assay of a more complete selection of acid-stable commercially
available cysteine protecting groups to these DTNP deprotection
conditions. This selection of Cys S-protectants, listed in Table 1,
have traditionally required very harsh and/or toxic conditions to
effect their removal [8–17]. We present the results of these efforts
to illustrate the utility of these comparatively more gentle condi-
tions of deprotection.
tetramethyluronium hexafluorophosphate (HBTU) were pur-
chased from RS Synthesis (Louisville, KY, USA). 2-(7-Aza-1H-benzo-
triazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) was purchased from Oakwood Products (Jackson Hole,
WY, USA). CLEAR-OX resin was purchased from Peptides Interna-
tional (Louisville, KY, USA). 2,2′-Dithiobis-5-nitropyridine (DTNP),
thioanisole, and all other reagents were purchased from Sigma-
Aldrich (Milwaukee, WI, USA). Native apamin was purchased
from American Peptide Company (Sunnyvale, CA, USA).
In addition to exploring the deprotection profiles of these Cys
derivatives on a small test peptide, we investigated the potential
for stepwise deprotection/disulfide-closure in model oxytocin
systems in which both cysteines were S-protected with identical
blocking motifs corresponding to the acid-stable blocking groups
in Table 1. In order to strongly illustrate the utility of this method
toward stepwise disulfide formation, we utilized a two-disulfide
apamin template bearing differentially protected orthogonal
Cys pairings that were iteratively deprotected and cyclized. An
understanding of the scope and limitations of these deprotec-
tion/cyclization procedures would be an important resource for
application to the design of new and more complex disulfide-
containing peptides.
Peptide Syntheses
All peptides were synthesized on a 40 mmole scale using 2-chlorotrityl
chloride resin (1.01 mmol/g loading). A SYMPHONY multiple pep-
tide synthesizer (Protein Technologies Inc., Tucson, AZ, USA) was
used to construct the peptide sequences via Fmoc protocol.
Double coupling using 1 : 3 HATU/HBTU activation was employed
for peptide elongation. A typical single coupling procedure was
as follows: 20% piperidine/DMF (2 Â 6 min); DMF washes
(6 Â 30 s); 5 equiv. Fmoc amino acid and HBTU in 0.4 M NMM/
DMF (2 Â 30 min); DMF washes (3 Â 30 s). Cleavage of peptides
from their resins was accomplished through treatment of the
resin with 94 : 2 : 2 : 2 TFA/TIPS/H2O/anisole for 2 h. Following
filtration of the resin, the cleavage supernatant was evaporated
to one tenth its original volume, followed by precipitation of the
crude peptide into cold anhydrous diethyl ether.
Materials and Methods
Materials
High-Pressure Liquid Chromatography
N,N-dimethylformamide, HPLC-grade acetonitrile, and trifluoroacetic
acid were purchased from Fisher Scientific (Pittsburgh, PA, USA).
Fmoc-Cys(Acm)-OH, Fmoc-Cys(Mob)-OH, Fmoc-Cys(StBu)-OH, Fmoc-Cys
(tBu)-OH, Fmoc-Glu(OtBu)-Thr(ΨMe,Mepro)-OH, and 2-chlorotrityl chlo-
ride resin were purchased from Novabiochem (San Diego, CA, USA).
All other Fmoc amino acids and O-benzotriazole-N,N,N′,N′-
High-pressure liquid chromatography analysis was done on a
Shimadzu analytical HPLC system with LC-10 AD pumps, SPD-
10A UV-Vis detector, and SCL-10A controller using a SymmetryW
C18-5 mm column from Waters (4.6 Â 150 mm). Aqueous and or-
ganic phases were 0.1% TFA in water (Buffer A) and 0.1% TFA in
HPLC-grade acetonitrile (Buffer B), respectively. Beginning with
Table 1. Representative selection of common cysteine S-protectants.
Traditional
Removal Conditions
Traditional
Removal Conditions
Name
Structure
Name
Structure
Na/liq. NH3
(ref. 8)
95% TFA
(ref. 14)
Bzl
Trt
R = H
R = H
50% HF/Anisole
(ref. 9)
1% TFA/DCM
(ref. 15)
Meb
Mmt
R = CH3
R = OCH3
AgOTf/TFA/thioanisole
(ref. 10)
Mob
R = OCH3
1% TFA/DCM
(ref. 16)
Xan
Hg(OAc)2
(ref. 11)
Acm
tBu
Hg(OTf)2
(ref. 12)
7% TFA/DCM
(ref. 17)
Tmob
P(Ph)3
(ref. 13)
StBu
Bzl = benzyl; Meb = 4-methylbenzyl; Mob = 4-methoxybenzyl; Acm = acetamidomethyl; tBu = tert-butyl; StBu = tert-butylmercapto-; Trt = trityl; Mmt = 4-
methoxytrityl; Xan = Xanthenyl; Tmob = 2,4,6-trimethoxybenzyl.
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DTNP AS A CYSTEINE DEPROTECTION REAGENT
100% Buffer A, a 1.4 ml/min gradient elution increase of 1%
Buffer B/min for 50 min was used for all analytical peptide chro-
matograms. Peptide signals were detected at both 214 and
254 nm. Preparative HPLC purification was carried out on a
Shimadzu preparatory HPLC system utilizing LC-8A pumps, an SPD-
10A UV-Vis detector, and an SCL-10A controller. A Waters Symmetry-
Prep C18 preparatory column (7 mm pore size, 1900 Â 150 mm)
was utilized in these separations. Beginning with 100% Buffer A, a
17 ml/min gradient elution increase of 1% Buffer B/min for 50 min
was used for all preparative chromatograms.
DTNP Deprotection Assay Conditions for VTGGC(X)A Test Peptides
One-milligram (~1.7 mmol) aliquots of VTGGC(X)A test peptides
1–6 were dissolved in 200 mL of either 100% TFA or 2% thioani-
sole/TFA to a final concentration of ~ 8.5 mM. Each of these solu-
tions was incubated with differing quantities of DTNP
corresponding to 1.1eq. (10 mM), 3.3 eq. (30 mM), 6.7 eq. (60 mM),
11 eq. (100 mM), and 15 eq. (137 mM) with agitation at 25 ꢀC for
1 h. At the end of this time, cold diethyl ether was added to each
reaction, and the crude precipitated product was isolated by cen-
trifugation. After drying the pellets, the crude isolates were dis-
solved in 10% ACN/H2O buffered to 0.1% TFA and subjected to
analytical HPLC analysis.
Mass Spectrometry
Matrix-assisted laser desorption ionization/Time-of-flight (MALDI-
TOF) mass spectra were collected on a Voyager DE-Pro instrument
under positive ionization and in reflectron mode. All samples
were run using a matrix of 10 mg/ml 2,5-dihydroxybenzoic
acid, vacuum-dried from a solution of 1 : 1 H2O/Acetonitrile
(ACN) buffered to 0.1% TFA. Mass spectra of all peptide inter-
mediates and final products are reported in the Supporting In-
formation for this manuscript.
DTNP Deprotection and Disulfide Formation in Oxytocin Test
Peptides
Two-milligram (~1.7 mmol) aliquots of each di-protected oxytocin
peptide 7–10 were dissolved in 200 mL of 2% thioanisole/TFA to
a final concentration of ~ 8.5 mM. Each of these solutions was in-
cubated with 20 eq. (183 mM) of DTNP with agitation at varying
Table 2. DTNP deprotection parameters and results on model
Protecting group P
Peptide synthetic yielda
Deprotection conditions
Eq. (mM) DTNP
Reaction
%
time/temperature
Deprotection/cyclization
(measured by HPLC)b
Bzl (1)
61%
57%
58%
43%
50%
45%
15 (137)
15 (137)
3 (27)
1 h/25 ꢀC
1 h/25 ꢀC
1 h/25 ꢀC
1 h/25 ꢀC
1 h/25 ꢀC
1 h/25 ꢀC
0
Meb (2)
Mob (3)
Acm (4)
tBu (5)
16
100
86
15 (137)
15 (137)
1 (9)
92
StBu (6)
100
Mob (7)
Acm (8)
tBu (9)
32%
33%
40%
34%
20 (183)
20 (183)
20 (183)
20 (183)
3 h/37 ꢀC
8 h/50 ꢀC
3 h/50 ꢀC
8 h/50 ꢀC
100
45
100
NA
100
95
100
100
StBu (10)
Mob (11)
Acm (12)
tBu (13)
49%
67%
76%
32%
58%
46%
20 (73)
20 (73)
20 (73)
3 h/37 ꢀC
8 h/50 ꢀC
8 h/50 ꢀC
100
100
100
100
100
100
(14)
36%
20 (73)
8 h/50 ꢀC
100
100
77
100
aFor apamin constructs 11–13, the first value corresponds to synthetic yield of uncyclized peptide, and the second value corresponds to isolated yield of
CLEAROX-mediated first disulfide closure (illustration shown).
bFor apamin constructs 11–14, the first value corresponds to the effectiveness (determined by HPLC) of DTNP deprotection, and the second value corre-
sponds to HPLC-derived effectiveness for DTT-cyclization of bis-Npys intermediate.
J. Pept. Sci. 2012; 18: 1–9
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SCHROLL, HONDAL AND FLEMER
temperatures and reaction times as listed in Table 2. At the end of
the reaction time, cold diethyl ether was added to each reaction,
and the crude precipitated product was isolated by centrifuga-
tion. After drying the pellets, the crude isolates were dissolved
in 2–3 mL 100 mM NH4HCO3 in 9 : 1 H2O/ACN [pH 7.5] to yield a
final peptide concentration of 1–2 mM. To a vigorously stirred solu-
tion of each of these intermediates, 1 eq. of dithiothreitol (DTT) dis-
solved in 500 mL of the same buffer was added in one portion, and
the solution was allowed to stir for an additional 5 min. Following
analytical HPLC analysis of the mixture, the entire reaction con-
tents were injected onto the preparative HPLC and the disulfide-
containing native oxytocin was isolated by lyophilization of the
fractions containing the desired product [m/z: 1010.3 (M + H)].
Stepwise DTNP Deprotection and Disulfide Formation in
Orthogonally Protected Apamin Test Peptide 14
Twenty milligrams (~8.5 mmol) of C1,11(StBu)-C3,13(tBu) apamin
peptide 14 was dissolved in 700 mL of TFA to a final concentra-
tion of ~ 12.1 mM. This solution was incubated with 54 mg
(20 eq.) DTNP under time and temperature conditions as listed in
Table 2. At the end of this time, cold diethyl ether was added to
each reaction, and the crude precipitated product was isolated
by centrifugation. Following drying of the pellet, the crude isolate
was dissolved in 10 mL 100 mM NH4HCO3 in 9 : 1 H2O/ACN [pH
7.5] yielding a final peptide concentration of 1–2 mM and treated
with 1 eq. DTT as previously described to yield the C3,13-cyclized,
C1,11(StBu)-protected intermediate [m/z: 2207.6 (M + H)] that was,
following analytical HPLC analysis, purified via the injection of the
entire reaction contents onto the preparative HPLC. Fractions
containing the desired product were frozen to À80 ꢀC
and lyophilized. Seven milligram (3.17 mmol) C3,13-cyclized,
First Disulfide Formation in Apamin Test Peptides 11–13
To install the first disulfide connectivity, 10 mg (~4.5 mmol) of
each C1,11-(X), C3,15-(SH) apamin 11–13 were dissolved in 5 mL
100 mM NH4HCO3 in 9 : 1 H2O/ACN [pH 7.5] and gently agitated
with 0.215 g (10 eq.) CLEAR-OX resin (0.21 mmol/g) [18] for 3 h.
The solution containing the peptide was filtered, and the resin
was washed with 3 Â 3 mL 9 : 1 H2O/ACN. All of the washes were
combined with the original filtrate and the solution was frozen to
À80 ꢀC and lyophilized. Following lyophilization of the crude iso-
late, preparative HPLC purification and subsequent lyophilization
was carried out to afford the single disulfide-bonded intermedi-
ate [11 m/z: 2271.6 (M + H); 12 m/z: 2173.5 (M + H); 13 m/z:
2143.5 (M + H)].
C
1,11(StBu)-protected intermediate was dissolved in 500 mL of
2% thioanisole/TFA, followed by addition of 20 mg (20 eq.) DTNP
under time and temperature conditions as listed in Table 2. At the
end of this time, Et2O-workup and closure of the remaining disul-
fide connectivity via treatment with 1 eq. DTT was carried out as
previously described to afford the native apamin construct [m/z:
2029.5 (M + H)]. The identity of the synthetic apamin was
confirmed by coinjection with an authentic sample (see Support-
ing Information).
Results and Discussion
Because it was a goal of this research to examine the deprotec-
tion profiles of the common Cys S-protectants shown in Table 1
via this DTNP-mediated approach, we first needed to prioritize
the large number of these protecting groups into an assemblage
that would illustrate a benefit to their use in SPPS. As such, we im-
mediately disregarded all acid-labile blocking groups because of
the TFA-mediated conditions of this protocol. Although the Acm
and Mob functionality had been investigated by us previously [6],
we chose to reproduce that data along with that from the depro-
tection assays of the heretofore untested Cys S-protectants in or-
der to directly compare and contrast all of their deprotection
profiles.
As illustrated in Figure 1, the general protocol for Cys S-
deprotection in the test systems involved the incubation of the
protected Cys-containing peptide with DTNP in a 2% thioanisole/
TFA (or neat TFA) solvent system under time and temperature
constraints specific for the test sequence. During the incubation
with DTNP, the Cys S-protectant is removed and substituted by
the 2-(5-nitropyridyl) (Npys) group derived from DTNP fragmenta-
tion. The reaction was then quenched via precipitation of the entire
contents into cold diethyl ether. After isolation of the precipitate via
DTNP Deprotection and Second Disulfide Formation in
Apamin Test Peptides 11–13
Five milligrams (~2.3 mmol) of each disulfide-bonded intermedi-
ate 11–13 was dissolved in 500 mL of 2% thioanisole/TFA to a fi-
nal concentration of ~ 4.6 mM. Each of these solutions was
incubated with differing quantities of DTNP under varying time
and temperature conditions dependent upon the identity of
the protecting group (see Table 2). At the end of this time, cold
diethyl ether was added to each reaction, and the crude precipi-
tated product was isolated by centrifugation. Following drying of
the pellets, the crude isolates were dissolved in 2–3 mL 100 mM
NH4HCO3 in 9 : 1 H2O/ACN [pH 7.5] to yield a final peptide con-
centration of 1–2 mM and treated with 1 eq. DTT as previously de-
scribed to yield the 2-disulfide-containing native apamin [m/z:
2029.5 (M + H)] that, following analytical HPLC analysis, was puri-
fied via the injection of the entire reaction contents onto the pre-
parative HPLC. Fractions containing the desired product were
frozen to À80 ꢀC and lyophilized. The identity of the synthetic
apamin was confirmed by coinjection with an authentic sample
(see Supporting Information).
Figure 1. General DTNP deprotection overview showing conversion to 2-(5-nitropyridyl) [5-Npys] intermediate derived from DTNP fragmentation. Fac-
ile conversion to the cysteine thiol was achieved by treatment of the crude deprotected peptide isolate with excess thiol reductant such as DTT.
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DTNP AS A CYSTEINE DEPROTECTION REAGENT
centrifugation, the crude material was either maintained as its Npys
conjugate or (in the case of multiple-thiol-containing systems)
treated with one equivalent of DTT in order to induce collapse into
the desired disulfide.
results). Also, we were interested in using this deprotection ap-
proach in tandem with disulfide formation. To address these
issues and to illustrate a broader understanding of the utility of
this method, three peptide systems were constructed encom-
passing a wide diversity of length, sequence, and disulfide archi-
tecture. The first system bore the identical sequence (VTGGCA) as
the small test peptide previously studied on Acm- and Mob-pro-
tected cysteines [6], except that in this case additional peptides
were built bearing S-protection corresponding to the acid-stable
Cys derivatives outlined in Table 1. A second oxytocin tem-
plate [19] was constructed in order to merge a standard DTNP
deprotection of identically protected cysteine pairs with subse-
quent disulfide formation to yield the native cyclized sequence.
Lastly, an apamin peptide template [20] was designed to high-
light the use of this deprotection methodology in an orthogonal
fashion within a two disulfide-containing system.
The mechanism for this deprotection frequently required
thioanisole as an additive, as most of the S-protecting groups stud-
ied in this investigation are stable in its absence. As previously
reported [6] and summarized in Figure 2A, the assumed reactive
intermediate in the deprotection sequence is the trivalent sulfo-
nium thioanisole-Npys conjugate derived from attack of the
thioanisole sulfur on the electron-deficient disulfide of DTNP. This
intermediate, when in proximity to the protected cysteine,
induces attack upon the very reactive sulfur within the conjugate,
forming a trivalent sulfonium intermediate at the cysteine
residue. This transitory species ultimately collapses into its
corresponding Npys conjugate following attack of a scavenger
species (Figure 2B).
The linear syntheses of the VTGGCA and oxytocin sequences
were carried out in a routine fashion with no difficulties. Synthe-
ses of the apamin sequences were surprisingly challenging, how-
ever, given the presumed ease with similar systems whose
construction has been described in the literature [21]. Early
attempts at synthesis of this sequence met with crude product
profiles fraught with deletion sequences, as evidenced by HPLC.
These deletion-prone syntheses were dismayingly reproducible,
even when using very low-loading resins and employing very
high-end coupling conditions (i.e. HATU). The key to overcoming
this obstacle was finally discovered to be the use of a Glu7-Thr8
pseudoproline dipeptide sequence [22] in order to disrupt unde-
sired secondary-structure formation during construction. The in-
stallation of this specialized dipeptide during the synthesis of
the apamin constructs proved successful in overcoming the
majority of deletion problems inherent in the linear sequence.
DTNP-deprotection assays of the VTGGCA test systems were car-
ried out on equimolar aliquots of protected peptide at increasing
intervals of DTNP concentration in both neat TFA and 2% thioani-
sole/TFA. Following ether precipitation of each reaction, comparison
of HPLC chromatogram peak areas of the crude deprotection isolates
allowed for deblocking efficiency to be plotted as a function of
The basis for determination of the robustness of the acid-stable
protecting groups shown in Table 1 was the quantity of DTNP re-
quired for removal of each one. In our previous report, these
amounts were specified as the number of equivalents of DTNP
added to a particular volume of 2% thioanisole/TFA, because of
the fact that some species required only stoichiometric amounts
of DTNP to effect complete protecting group removal. For the
present study, we thought it important to measure actual con-
centration (mM) of DTNP, as it illustrates a more realistic kinetic
measure of deprotection effectiveness, especially when studying
the deprotection profile of the more robust protecting groups.
Furthermore, an absolute concentration value better expresses
differences in effective concentration brought about by variations
in solution volume when identical stoichiometric amounts of
DTNP are used in assays.
Our intention was to study the effectiveness of this deprotec-
tion methodology from a number of different standpoints. First,
it had become clear since our initial publication that the depro-
tective potential of this process was heavily dependent upon
specific amino acid sequence, peptide length, and cysteine place-
ment within the sequence (Flemer S, Hondal RJ, unpublished
Figure 2. Putative mechanism of DTNP-mediated deprotection. (A) Formation of activated DTNP-thioanisole intermediate. (B) Cysteine deprotection
brought about by initial attack of cysteine sulfur on activated intermediate to form trivalent sulfonium intermediate, followed by collapse into the
5-Npys intermediate by scavenging of the protecting group fragment.
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SCHROLL, HONDAL AND FLEMER
Figure 3. Graphic representation of the effectiveness of DTNP-mediated deprotection in VTGGCA test peptide systems (1–6) containing a selection of
cysteine S-protectants, showing comparisons of deprotection effectiveness in the presence and absence of thioanisole.
increasing DTNP concentration for each protecting group assayed
(Figure 3). In the case of these simple test sequences, the depro-
tected species were measured as their 5-Npys conjugates.
As illustrated in Figure 3, a diverse spectrum of applicability
was found for this deprotection methodology toward these cys-
teine S-protectants. The benzyl-templated series show differing
extents of lability, highly dependent upon the identity of the
electron-donating substituent at the para position on the phenyl
ring, with effectiveness of deprotection noted as Mob > Meb >
Bzl. The Bzl functionality showed no lability whatsoever even at
higher concentrations of DTNP, whereas the Meb group demon-
strated only partial (~16%) removal. In the presence of thioanisole,
the Mob group was completely removed when treated with ~2eq.
(20 mM) DTNP. However, in the absence of thioanisole, the Mob
functionality was much more robust, showing only partial
removal, even at greatly increased DTNP concentrations.
Deprotection assays of the remaining Cys S-protectants revealed
additional instances in which thioanisole played a vital role in
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DTNP AS A CYSTEINE DEPROTECTION REAGENT
deprotection efficiency as well as those in which it did not. The S-
deprotection profile of the tBu group, for instance, showed equal
efficiency to the DTNP conditions whether thioanisole was used
or not. This finding stands to reason because of the existing effi-
cient Npys-Cl tBu-removal vector for cysteine [23], based upon
both reagents’ installation of the identical electron-deficient 5-
Npys moiety onto the Cys thiol. In contrast to the thioanisole-in-
dependence of the tBu deprotection protocol, the DTNP-lability
of the Acm and StBu functionalities were more highly thioani-
sole-dependent. Acm deprotection followed the general trend
as that of Mob, although it was found to be much more robust.
The DTNP deprotection profile of the StBu group was quite re-
markable in that there existed an absolute requirement for thioa-
nisole. As illustrated in Figure 3, StBu protection was exceedingly
robust in the absence of thioanisole, showing no lability even at
elevated DTNP concentrations. However, in the presence of
thioanisole, complete StBu deprotection was carried out at stoi-
chiometric DTNP concentrations. Indeed, the StBu group high-
lighted the squarest orthogonality profile yet observed using
this methodology.
It was found that the previously utilized DTNP conditions did
not translate to the expected extent of deprotection in the case
of oxytocin test sequences 7–10. In fact, even at higher DTNP con-
centrations, MALDI analysis indicated that although one Cys was
consistently converted to its corresponding 5-Npys conjugate,
the other partner was sluggish to deprotect and subsequently
stalled, showing incomplete global deprotection under the previ-
ously used conditions (data not shown). As a result, higher DTNP
concentrations with increased temperature and time were re-
quired to effect dual deprotection of these constructs in most
instances. It was found that the ease of bis-deprotection followed
the trend Mob > tBu > StBu > Acm, with full deprotection of the
bis-Acm pair unattainable. Surprisingly, although the StBu protec-
tion was most easily removed on the single cysteine containing
test peptide 6, it was one of the more robust protecting groups
in the bis-Cys containing oxytocin template.
less than 1% formation of the cyclized native peptide. We envi-
sioned that it might be possible to induce disulfide cyclization
if, after isolation of the bis-Npys intermediate, one equivalent of
thiol reductant (i.e. DTT) was added to a NH4HCO3-buffered aque-
ous solution of this intermediate to carry out reduction of one of
the Npys moieties, leading to spontaneous cyclization of the lib-
erated cysteine thiol upon its Npys-conjugated partner to afford
the native oxytocin species. After some optimization, we settled
on a method of incubating the crude deprotection isolate in a
100 mM NH4HCO3 buffer to a final optimal peptide concentration
of 1 mM. To this briskly stirred mixture, a 1 eq. bolus of DTT was
added followed by immediate HPLC analysis of the resulting
deep yellow solution. Identical HPLC profiles from various time
points after the initial addition of DTT indicated that the collapse
of the hemi-reduced intermediate into the native oxytocin was
virtually instantaneous (Figure 4).
Because the apamin constructs were templates for highlighting
this method’s utility in stepwise disulfide construction, the DTNP
deprotection procedure was merely a component of a larger pro-
cess. As illustrated in Table 2, peptides 11–13 were designed
to have two differentially protected cysteine pairs. The Cys3,15
pair, possessing trityl S-protection during synthesis, provide
native thiol functionality upon peptide cleavage. Alternatively,
the Cys1,11 pair was designed to bear the more robust protection
protocol (Mob, Acm, tBu) whose removal was to be effected by
the DTNP-mediated process. The cyclization of the Cys3,15 free
thiol pair was achieved by incubation of an aqueous solution of
each peptide with CLEAR-OX resin [18]. Following isolation and
purification of this intermediate, the DTNP deprotection protocol
was then carried out as previously described. In addition to the
protected Cys pairs requiring different concentrations of DTNP
to effect full deprotection, apamin constructs 11–13 required el-
evated reaction temperatures of 37–50 ꢀC and longer reaction
times in order to drive the dual deprotection to completion, sim-
ilar to those required for the bis-deprotection of oxytocin systems
7–10, except that in this case the bis-Acm pair on the apamin
construct could be fully deprotected. This finding again high-
lighted the differences in deprotection effectiveness using this
methodology when applied to different peptide sequences.
Once the DTNP deprotection chemistry had been carried
out on each apamin template, HPLC and MALDI data again
It was hoped that in carrying out these dual DTNP deprotections
on the oxytocin sequences, spontaneous disulfide formation might
occur because of the highly favorable disposition of the two cyste-
ine residues within the sequence. It was found, however, that the
species that emerged in all cases was the bis-Npys adduct, with
Figure 4. Stepwise HPLC comparison of the deprotective profile of oxytocin systems 7–10 (bis-Acm system used as example). Chromatogram on left
shows bis-Acm-protected oxytocin peptide (1). Middle chromatogram shows crude HPLC profile following treatment with DTNP/thioanisole of bis-Npys
oxytocin intermediate (2). Chromatogram on right is crude HPLC profile following treatment with 1 eq. DTT, showing peaks for free 5-Npys moiety (3)
and cyclized native oxytocin (4).
J. Pept. Sci. 2012; 18: 1–9
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SCHROLL, HONDAL AND FLEMER
suggested that the crude isolates consisted exclusively of the
bis-Npys species. In similar fashion to the previous disulfide clo-
sure on oxytocin models 7–10, the addition of 1 eq. DTT to each
peptide dissolved in aqueous buffer induced the singular
collapse of these intermediates into native the apamin structure
(Figure 5a). Care had to be taken in these apamin conversions,
however, as we found that initial peptide isolate concentrations
exceeding 2 mM began to show competing intermolecular
disulfide formation (data not shown). Coinjection of the experi-
mentally derived apamin constructs with an authentic sample
yielded a single peak without splitting or shouldering, confirming
that disulfide scrambling had not occurred (see Supporting
Information).
As a final testament to the selectivity of this approach and
to its applicability toward stepwise disulfide formation, an
apamin-based model system was constructed bearing tBu-
protection on the Cys3,15 pair and StBu-protection at the Cys1,11
pair (14). Because these two protecting groups were shown to
be orthogonal in the absence of thioanisole, a two-step approach
toward sequential disulfide formation was carried out (Figure 5B).
The peptide was carried through one iteration of (tBu) deprotec-
tion/cyclization via treatment with DTNP/TFA in the absence of
thioanisole followed by treatment of the crude isolate in
100 mM ammonium bicarbonate with 1 eq. DTT. HPLC and MALDI
analysis of the resulting solution showed that both of the tBu pro-
tectants were indeed removed in the presence of the StBu
groups and the Cys3,15 disulfide was selectively formed [m/z:
2207.7 (M + H)]. Following isolation and purification of the
hemi-cyclized intermediate, further treatment with DTNP (this
time in the presence of thioanisole) removed both StBu protec-
tants, and the second disulfide bond was formed as previously
described. Most likely because of the underlying architecture
of construct 14, some difficulties were encountered in the DTT-
cyclization step following the first (tBu) deprotection. It was
found that, in addition to the desired single-disulfide inter-
mediate produced by DTT-mediated collapse of the bis-Npys in-
termediate, a minor yet significant amount of native apamin
was also present in the crude reduction mixture. This was likely
the result of unintentional over-reduction of the intermediate,
in which excess DTT partially reduced the StBu protecting
Figure 5. (A) Stepwise HPLC comparison of the deprotective profile of apamin systems 11–13. Chromatogram on left shows C1,11(protected)-C3,13(SH)
apamin peptide (bis-Acm system used as example) (1). Middle Chromatogram illustrates crude HPLC profile following treatment with CLEAR-OX resin,
showing peak for disulfide-bonded intermediate (2). Chromatogram on right is crude HPLC profile following treatment with DTNP/thioanisole followed
by 1 eq. DTT, showing peaks for free 5-Npys moiety (3) and cyclized native apamin (4). (B) Stepwise HPLC comparison of the deprotective profile of StBu/
tBu apamin system 14. Chromatogram on left shows StBu/tBu-protected apamin peptide (1). Middle chromatogram illustrates crude HPLC profile fol-
lowing treatment with DTNP and 1 eq. DTT, showing peaks for free 5-Npys moiety (2), native apamin (3), and desired cyclized StBu intermediate (4).
Chromatogram on right is crude HPLC profile of purified cyclized StBu intermediate following treatment with DTNP/thioanisole followed by 1 eq.
DTT, showing peaks for free 5-Npys moiety (2) and cyclized native apamin (5).
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J. Pept. Sci. 2012; 18: 1–9

DTNP AS A CYSTEINE DEPROTECTION REAGENT
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groups still present within the peptide framework and
subsequent collapse of the apamin construct into its conforma-
tionally favored native structure may have resulted. Because it is
well-known that Cys(StBu) is readily reduced with exogenous
thiol at slightly basic conditions, this over-reduction phenome-
non is not surprising.
6 Harris KM, Flemer S, Hondal RJ. Studies on deprotection of cysteine
and selenocysteine side-chain protecting groups. J. Pept. Sci. 2007;
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9 Erickson BW, Merrifield RB. Acid stability of several benzylic protecting
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10 Tamamura H, Otaka A, Takada W, Terakawa Y, Yoshizawa H, Masuda
M, Ibuka T, Murakami T, Nakashima H, Waki M, Matsumoto A,
Yamamoto N, Fujii N. Solution-phase synthesis of an anti-human im-
munodeficiency virus peptide, T22 ([Tyr5,12, Lys7]-polyphemusin II),
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during trimethylsilyl trifluoromethanesulfonate deprotection. Chem.
Pharm. Bull. 1995; 43: 12–18.
11 Veber DF, Milkowski JD, Varga SL, Denkewalter RG, Hirschmann R.
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12 Nishimura O, Kitada C, Fujino M. New method for removing the S-p-
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ric trifluoroacetate. Chem. Pharm. Bull. 1978; 26: 1576–1585.
13 Moroder L, Gemeiner M, Goehring W, Jaeger E, Thamm P, Wunsch E.
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14 Kellenberger C, Hietter H, Luu B. Regioselective formation of the three
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15 Barlos K, Gatos D, Hatzi O, Koch N, Koutsogianni S. Synthesis of
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An expansion on previous work pertaining to the effective-
ness of DTNP/TFA deprotection on two common cysteine S-
protecting groups has been carried out. Specifically, the
acid-stable protecting groups listed in Table 1 were assayed
to determine their respective lability to these same conditions
in both the presence and the absence of thioanisole. DTNP assays
carried out on Cys test peptides bearing these six protecting
groups in the presence as well as the absence of thioanisole
showed a wide diversity of deprotective effectiveness. In some
cases, in particular that of the StBu-protected Cys peptide, thioa-
nisole additive accelerated the deprotection significantly. These
protecting group lability trends, once determined, were applied
to more complex peptide models bearing multiple Cys residues,
carrying out protecting group removal in tandem with stepwise
disulfide closure.
The continued development of new protecting groups or
application of new conditions for more facile removal of exist-
ing protecting groups is a crucial consideration when design-
ing chemical syntheses of increasingly complex peptides. Be-
cause of the high reactivity of its side-chain thiol, blocking
protocol for cysteine has enhanced importance in approaches to-
ward iterative assembly of multiple disulfide-containing peptide
systems. The method expanded upon here adds an essential,
milder deprotection vector for many commercially available cys-
teine protectants. It is hoped that this methodology can be ap-
plied in greater detail to amino acid constructs similar in
reactivity to cysteine (i.e. selenocysteine) but whose current pro-
tection scheme is limited by architecture and available deprotec-
tion methodologies.
16 Han Y. Barany G. Novel S-xanthenyl protecting groups for cysteine and
their applications for the Na-9-Fluorenylmethyloxycarbonyl (Fmoc)
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17 Munson M.C. Garcia-Echeverria C. Albericio F. Barany G. S-2,4,6- tri-
methoxybenzyl (Tmob): a novel cysteine protecting group for the N-
a-(9- fluorenylmethoxycarbonyl) (Fmoc) strategy of peptide synthesis.
J. Org. Chem. 1992; 57: 3013–3018.
HPLC chromatograms and MALDI mass spectra of all peptide
intermediates and final products are contained in the Supporting
Information section associated with this article.
18 Darlak K, Wiegandt-Long D, Czerwinski A, Darlak M, Valenzuela F,
Spatola AF, Barany G. Facile preparation of disulfide-bridged peptides
using the polymer-supported oxidant CLEAR-OX. J. Peptide Res. 2004;
63: 303–312.
Acknowledgements
These studies were supported by National Institutes of Health
grant GM094172 to RJH and Vermont Genetics Network Grant
P20 RR16462 to ALS.
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tator of life. Prog. Neurobiol. 2009; 88: 127–151.
20 Habermann E. Apamin Pharmacol. Ther. 1984; 25: 255–270.
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natural cystine/selenocystine connectivities. Biopolymers (Peptide Science)
1999; 53: 550–564.
22 Garcia-Martin F, White P, Steinauer R, Cote S, Tulla-Pucche J, Albericio F.
The synergy of ChemMatrix resin and pseudoproline building blocks
renders RANTES, a complex aggregated chemokine. Biopolymers
(Peptide Science) 2006; 84: 566–575.
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