The use of 2,2′-dithiobis(5-nitropyridine) (DTNP) for deprotection and diselenide formation in protected selenocysteine-containing peptides

Article abstract of DOI:10.1002/psc.1430

In contrast to the large number of sidechain protecting groups available for cysteine derivatives in solid phase peptide synthesis, there is a striking paucity of analogous selenocysteine Se-protecting groups in the literature. However, the growing interest in selenocysteine-containing peptides and proteins requires a corresponding increase in availability of synthetic routes into these target molecules. It therefore becomes important to design new sidechain protection strategies for selenocysteine as well as multiple and novel deprotection chemistry for their removal. In this paper, we outline the synthesis of two new Fmoc selenocysteine derivatives [Fmoc-Sec(Meb) and Fmoc-Sec(Bzl)] to accompany the commercially available Fmoc-Sec(Mob) derivative and incorporate them into two model peptides. Sec-deprotection assays were carried out on these peptides using 2,2′-dithiobis(5-nitropyridine) (DTNP) conditions previously described by our group. The deprotective methodology was further evaluated as to its suitability towards mediating concurrent diselenide formation in oxytocin-templated target peptides. Sec(Mob) and Sec(Meb) were found to be extremely labile to the DTNP conditions whether in the presence or absence of thioanisole, whereas Sec(Bzl) was robust to DTNP in the absence of thioanisole but quite labile in its presence. In multiple Sec-containing model peptides, it was shown that bis-Sec(Mob)-containing systems spontaneously cyclize to the diselenide using 1eq DTNP, whereas bis-Sec(Meb) and Sec(Bzl) models required additional manipulation to induce cyclization.

Full text of DOI:10.1002/psc.1430

Research Article
Received: 27 August 2011
Revised: 10 October 2011
Accepted: 14 October 2011
Published online in Wiley Online Library: 16 January 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.1430
The use of 2,2′-dithiobis(5-nitropyridine)
(DTNP) for deprotection and diselenide
formation in protected selenocysteine-
containing peptides
Alayne L. Schroll,a Robert J. Hondalb,c* and Stevenson Flemer Jr.c*
In contrast to the large number of sidechain protecting groups available for cysteine derivatives in solid phase peptide
synthesis, there is a striking paucity of analogous selenocysteine Se-protecting groups in the literature. However, the growing
interest in selenocysteine-containing peptides and proteins requires a corresponding increase in availability of synthetic
routes into these target molecules. It therefore becomes important to design new sidechain protection strategies for seleno-
cysteine as well as multiple and novel deprotection chemistry for their removal. In this paper, we outline the synthesis of two
new Fmoc selenocysteine derivatives [Fmoc-Sec(Meb) and Fmoc-Sec(Bzl)] to accompany the commercially available Fmoc-Sec
(Mob) derivative and incorporate them into two model peptides. Sec-deprotection assays were carried out on these peptides
using 2,2′-dithiobis(5-nitropyridine) (DTNP) conditions previously described by our group. The deprotective methodology was
further evaluated as to its suitability towards mediating concurrent diselenide formation in oxytocin-templated target pep-
tides. Sec(Mob) and Sec(Meb) were found to be extremely labile to the DTNP conditions whether in the presence or absence
of thioanisole, whereas Sec(Bzl) was robust to DTNP in the absence of thioanisole but quite labile in its presence. In multiple
Sec-containing model peptides, it was shown that bis-Sec(Mob)-containing systems spontaneously cyclize to the diselenide
using 1 eq DTNP, whereas bis-Sec(Meb) and Sec(Bzl) models required additional manipulation to induce cyclization. Copyright
© 2012 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: selenocysteine; protecting group; deprotection; DTNP; thioanisole; selenol
(Mob) group. Moreover, although Mob Se-protection is utilized
Introduction
on Sec derivatives bearing both Boc and Fmoc aN protection,
Selenocysteine (Sec, U) is the ‘21st’ proteinogenic amino acid be-
cause it shares three key properties with the other 20 common
amino acids: it has its own codon (UGA), it has its own unique
tRNA, and it is co-translationally inserted at the ribosome [1,2].
Heterologous expression and production of selenoproteins is dif-
ficult because of the complexity of the UGA recoding machinery
needed by the ribosome to translate UGA as a sense codon for
Sec instead of as a stop codon [3,4]. A potential solution for over-
coming the biological barriers for making Sec-containing pro-
teins and peptides is by using solid phase peptide synthesis
(SPPS). However, this route also presents its own set of problems
including racemization, deselenization, a sparse choice of Se-
protecting groups, and a lack of orthogonal methods for making
selenosulfide and diselenide bonds [5–7]. Here, we report some
advances in solving the latter two problems.
In contrast to the great number of sidechain S-protecting
groups for cysteine in SPPS [8,9], there is a scarcity of sidechain
Se-protecting groups for selenocysteine. With the exception of
some novel sidechain protection strategy being advanced for
Sec of late [10], the same general sets of benzyl-templated Sec
sidechain protection functionality have persevered unadvanced
for decades (Figure 1) [11–14]. In addition to the traditional
benzyl functionality, electron-releasing para substituents give rise
to the 4-methylbenzyl (Meb) group and the 4-methoxybenzyl
the Bzl and Meb blocking motifs have so far only been utilized
in Boc-protected Sec derivatives.
Traditionally, because of the robust nature of these benzyl-
type protective moieties, conditions of deprotection have relied
upon very harsh conditions such as the use of Hydroflouric Acid
(HF) [15], molecular iodine [5], and metallic sodium in ammonia
[11]. Recent research efforts in our laboratory have led to the dis-
covery of a set of deprotection conditions for various Cys and Sec
*
*
Correspondence to: Stevenson Flemer, Department of Chemistry, Cook
Physical Sciences Bldg., 82 University Place, University of Vermont, Burlington,
VT 05405, USA. E-mail: sflemer@uvm.edu
Robert J. Hondal, Department of Biochemistry, Room B413 Given Bldg, 89
Beaumont Ave, University of Vermont, Burlington, VT 05405, USA. E-mail:
Robert.Hondal@uvm.edu
a Department of Chemistry, Saint Michael’s College, One Winooski Park,
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 Chemistry, Cook Physical Sciences Bldg, University of Vermont,
82 University Place, Burlington, VT 05405, USA
J. Pept. Sci. 2012; 18: 155–162
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

SCHROLL, HONDAL AND FLEMER
Synthesis of Fmoc-Sec(X) Derivatives
Two procedures (1 and 2) were adapted with some modification
for the synthesis of Fmoc-Sec derivatives (see Figure 3).
Procedure 1
Figure 1. Inventory of traditional benzyl-templated Sec-protected SPPS
derivatives.
This procedure is similar to that previously described [18] with
some modification.
sidechain protecting groups that are much gentler than conven-
tional approaches [16]. This methodology, which has been opti-
mized for use with several Cys protecting groups [17], utilizes 2,
2′-dithiobis(5-nitropyridine) (DTNP) in a TFA/thioanisole cocktail
to effect chalcogen deprotection under much less harsh conditions
than traditionally possible (Figure 2). Although we previously
reported some limited data on the enhanced effectiveness of these
conditions towards Sec(Mob) deprotection [tenfold more effective
than the corresponding Cys(Mob) deprotection] [16], we report here
a comprehensive overview of the scope and of this deprotection
methodology towards the three major benzyl-templated Sec
sidechain blocking groups. Furthermore, because our laboratory
is optimized towards Fmoc peptide synthesis rather than Boc
synthesis, we needed to develop syntheses of the Fmoc Sec deriv-
atives bearing Meb and Bzl protection at the selenol sidechain,
compounds whose syntheses have not been published. The two
peptides in which these Fmoc Sec derivatives were installed were
carefully assayed as to their suitability to deprotection under
DTNP-mediated conditions as well as to their abilities to cyclize
into diselenide-containing targets.
Synthesis of H-Sec(X)-OH intermediates (2a–c)
In a 250-ml round-bottom flask equipped with a stirring bar un-
der inert N2 atmosphere, selenocystine 1 (2.8 g, 8.3 mmol) was
slurried in degassed 0.5 M aqueous NaOH solution (10 ml). So-
dium borohydride (1.9 g, 49.7 mmol) dissolved in degassed water
(20 ml) was added slowly using syringe to the selenocystine
slurry. After stirring for 30min at 0 ꢀC, excess borohydride was
quenched via dropwise addition of glacial acetic acid until all bub-
bling had ceased. Of the benzyl chloride, 16.6mmol of the benzyl
chloride was then added dropwise over 20 min, and the reaction
was stirred under N2 at 0 ꢀC for 2 h. Concentrated HCl (4ml) was
then slowly added, and the reaction was maintained at 4 ꢀC over-
night. The crude Se-protected derivative was then isolated via suc-
tion filtration and used in the next step without further purification.
Synthesis of Fmoc-Sec(X)-OH intermediates (3a–c)
6.2 mmol of Sec(X)-OH [where X = Mob (2a), Meb (2b), and Bzl
(2c)] was slurried in water (10 ml) in a 250-ml round-bottom flask
with stirring. After the slurry was brought to 0 ꢀC, triethylamine
(1.70 ml, 12.4 mmol) was added in one portion followed by
Fmoc-O-succinimide (2.3 g, 6.8 mmol) dissolved in acetonitrile
(10 ml) with stirring. The reaction was then allowed to proceed
for 2 h at room temperature, monitored using TLC. The reaction
was quenched by the addition of 1 N HCl (50 ml), and the product
was extracted with ethyl acetate (2 Â 75 ml). The organic portions
were combined and back-extracted with 1 N HCl (75 ml). The
organic extract was dried over MgSO4 and concentrated to a yel-
low oil under reduced pressure. The Fmoc-protected product was
then purified over silica gel using 0.1 : 2.9 : 97 AcOH/MeOH/DCM
as an eluent. The yields (over two steps) of protected Fmoc deriv-
atives were as follows: Fmoc-Sec(Mob)-OH (3a, 10%); Fmoc-Sec
(Meb)-OH (3b, 38%); and Fmoc-Sec(Bzl)-OH (3c, 80%).
Materials and Methods
Materials
N,N-dimethylformamide, HPLC-grade acetonitrile, and trifluoroacetic
acid were purchased from Fisher Scientific (Pittsburgh, PA, USA).
NovaPEGW HMPB resin and 2-chlorotrityl resin were purchased
from Novabiochem (San Diego, CA, USA). All standard Fmoc
amino acids and O-benzotriazole-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) were purchased from RS Synthesis
(Louisville, KY, USA). HATU was purchased from Oakwood Products
(Jackson Hole, WY, USA). DTNP, thioanisole, and all other reagents
were purchased from Sigma-Aldrich (Milwaukee, WI, USA).
Figure 2. Prior results on protected Cys-containing and Sec-containing peptides [16,17]. (A) DTNP methodology allows facile deprotection of a number
of common Cys S-protectants in the presence or absence of thioanisole. (B) Sec(Mob) deprotection required only catalytic amounts of DTNP in the
absence of thioanisole.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 155–162

DTNP FOR SELENOCYSTEINE DEPROTECTION
Figure 3. Synthetic routes to Sec derivatives 3a–c.
Procedure 2
Synthesis of benzyl-templated diselenides (5a–c)
This procedure is a methodology adapted and optimized from
previous similar syntheses of Dawson [19] and van der Donk
[20].
Mob-diselenide, Meb-diselenide, and Bzl-diselenide were synthe-
sized through a previously established procedure [20].
Synthesis of Fmoc-Sec(X)-OMe derivatives (6a–c)
Synthesis of Fmoc-Ser(Ts)-OMe (4)
Benzyl-templated diselenide (16.35 mmol) was dissolved in THF
(20 ml) in a 250-ml round-bottom flask, with a slow stream of
nitrogen continually purging the reaction vessel. After cooling
the reaction mixture to 0 ꢀC, NaBH4 (0.93 g, 24.53 mmol, 1.5 eq)
dissolved in 7 ml water (Proc. A) or 19.62 ml (19.62 mmol, 1.2 eq)
1.0 M LiBEt3H in THF (Proc. B) was added dropwise over 20 min
to the diselenide solution. Following the addition of borohydride
solution, the mixture was allowed to stir at 0 ꢀC for 15 min. At the
end of this time, Fmoc-Ser(Ts)-OMe (4) (5.40 g, 10.90 mmol,
0.67 eq) dissolved in 20 ml THF was added dropwise to the reac-
tion mixture. Following this addition, the reaction mixture was
allowed to stir at 0 ꢀC for 2 h. At the end of this time, AcOH was
added dropwise to the reaction (at 0 ꢀC) until no additional gas
evolution was observed. The entire reaction contents were then
poured into 150 ml water in a separatory funnel followed by
200 ml EtOAc. Following thorough mixing, the organic layer was
then separated and washed further with 2 Â 100 ml 0.5% HCl
solution. The organic phase was separated, dried over MgSO4,
and concentrated in vacuo to afford the crude Se-protected
methyl ester. The crude product was then purified over silica
gel (20–40% EtOAC/Hex) to yield the desired methyl ester com-
pounds 6a–c.
Fmoc-Ser-OH (25.0 g, 76.38 mmol) was dissolved in 300 ml DMF
and DIEA (13.9 ml, 84.02 mmol, 1.1 eq) was added in one por-
tion. The solution was cooled to 0 ꢀC and MeI (5.23 ml,
84.02 mol, 1.1 eq) was added slowly over 20 min. The mixture
was allowed to stir overnight at room temperature. The entire
mixture was then poured into 500 ml of ice-cold 1% aq. HCl.
To this slurry was added 400 ml EtOAc, and the new mixture
was thoroughly shaken. The organic layer was separated, and
the resulting aqueous layer was extracted once more with
200 ml EtOAc. The organic portions were combined and
extracted with 2 Â 500 ml 2% aq. NaHCO3. The resulting
organic layer was dried over MgSO4 and concentrated in vacuo
to yield Fmoc-Ser-OMe as a dense amorphous colorless solid
that was used without further purification directly in the next
step.
Fmoc-Ser-OMe from the previous step (assumed 76.38 mmol)
and TsCl (43.68 g, 0.229 mol, 3 eq) was brought to 0 ꢀC and pyri-
dine (135 ml) was added with stirring. This mixture was stirred
at 0 ꢀC for 7 h. At the end of this time, the mixture was poured
into 600 ml Et2O and extracted with water (2 Â 300 ml) and 1%
aq. HCl (2 Â 300 ml). The organic portion was dried over MgSO4
and concentrated in vacuo to afford crude Fmoc-Ser(Ts)-OMe,
which was purified batchwise over silica gel (20–40% EtOAc/
Hex) to yield the desired Fmoc-Ser(Ts)-OMe 4 as a colorless solid
(24.6 g, 65% for two steps).
Fmoc-Sec(Mob)-OMe (6a): (Proc. A: 3.32 g, 58%; Proc. B: 5.15 g,
90%) isolated as a colorless amorphous solid: melting point (mp)
76–77 ꢀC; [a]2D8 = À27.54ꢀ (c 2.0, EtOH); 1H NMR (500 MHz, CDCl3) d
7.74 (dd, J = 2.8 Hz, J = 7.5 Hz, 2H), 7.60 (t, J = 7.0 Hz, 2H), 7.57–7.62
J. Pept. Sci. 2012; 18: 155–162 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

SCHROLL, HONDAL AND FLEMER
(m, 2H), 7.27–7.33 (m, 2H), 7.18 (d, J = 8.3 Hz, 2H), 6.80 (d,
J = 8.5 Hz, 2H), 5.57 (d, J = 7.8 Hz, 1H), 4.66 (q, J = 7.4 Hz, 1H), 4.40
(d, J = 7.0 Hz, 2H), 4.23 (t, J = 6.9 Hz, 1H), 3.75 (s, 8H), 2.87–2.96
(m, 2H); 13C NMR (125 MHz, CDCl3) 171.3, 158.6, 155.6, 143.8,
143.7, 141.3, 130.4, 130.0, 127.7, 127.1, 125.1, 120.0, 114.0, 67.1,
4.63–4.70 (m, 1H), 4.37 (d, J = 4.2 Hz, 2H), 4.17 (t, J = 4.0 Hz, 1H),
3.73 (s, 2H), 2.86–2.97 (m, 2H); 13C NMR (125 MHz, CDCl3) 175.2,
155.9, 143.6, 143.4, 141.2, 138.3, 128.8, 128.5, 127.6, 127.0, 126.9,
124.9, 124.6, 119.9, 67.2, 54.4, 53.6, 46.9, 28.0, 25.2, 24.7 ppm; IR
(film) 1710 cmÀ1, 1514 cmÀ1, 737 cmÀ1; HRMS calculated for
C25H23NO4Se: 481.0794. Found: 481.0811 (M + H).
55.2, 53.8, 52.6, 47.1, 27.5, 25.6 ppm; IR (film) 1682 cmÀ1
,
1510 cmÀ1, 737 cmÀ1; high resolution mass spectrometry (HRMS)
calculated for C27H27NO5Se: 525.1054. Found: 525.1056 (M + H).
Fmoc-Sec(Meb)-OMe (6b): (Proc. A: 1.83 g, 33%; Proc. B: 4.83 g,
87%) isolated as a colorless amorphous solid: mp 109–110 ꢀC;
Peptide syntheses
All peptides were synthesized on a 60-mmole scale using
2-chlorotrityl chloride resin (1.51 mmol/g loading) as follows. A
Symphony™ multiple peptide synthesizer (Protein Technologies
Inc., Tucson, AZ, USA) was used for peptide syntheses via Fmoc
protocol. Double coupling using 1 : 3 HATU/HBTU activation
was employed for peptide elongation. A typical single coupling
procedure is as follows: 20% piperidine/DMF (2 Â 6 min); DMF
washes (6 Â 30 s); 5 eq each of standard Fmoc amino acid
and coupling agent in 0.2 M NMM/DMF (2 Â 40 min); DMF
washes (3 Â 30 s). Coupling of Fmoc-Sec derivatives was carried
out using three times equivalents each of amino acid deriva-
tive, HOAt, and DIC with 5-min preincubation. Cleavage of pep-
tides from their resins was accomplished through treatment of
the resin with 96 : 2 : 2 TFA/Triisopropylsilane (TIPS)/H2O for
1.5 h. Following filtration of the resin, the liquid cleavage mix-
ture was evaporated to one tenth its original volume in a
stream of nitrogen, followed by precipitation of the crude pep-
tide into cold anhydrous diethyl ether.
1
[a]2D8 = À31.07ꢀ (c 2.0, EtOH); H NMR (500 MHz, CDCl3) d 7.71 (d,
J = 7.5 Hz, 2H), 7.58 (t, J = 6.8 Hz, 2H), 7.32–7.39 (m, 2H), 7.26 (t,
J = 7.4 Hz, 2H), 7.12 (d, J = 7.7 Hz, 2H), 7.05 (d, J = 7.8 Hz, 2H), 5.64
(d, J = 8.0 Hz, 1H), 4.65 (q, J = 5.1 Hz, 1H), 4.38 (d, J = 7.0 Hz, 2H),
4.19 (t, J = 6.9 Hz, 1H), 3.71 (s, 2H), 3.69 (s, 3H), 2.82–2.87 (m, 2H),
2.26 (s, 3H); 13C NMR (125 MHz, CDCl3) 171.2, 155.5, 143.7, 143.5,
141.1, 136.4, 135.5, 129.2, 128.6, 127.5, 126.9, 124.9, 119.8, 67.0,
53.7, 52.4, 46.9, 27.5, 25.5, 20.9 ppm; IR (film) 1717 cmÀ1
1497 cmÀ1 762 cmÀ1
HRMS calculated for C27H27NO4Se:
509.1105. Found: 509.1106 (M + H).
,
,
;
Fmoc-Sec(Bzl)-OMe (6c): (Proc. A: 4.48 g, 83%; Proc. B: 4.86 g,
90%) isolated as a light yellow amorphous solid: mp 70–72 ꢀC;
[a]2D8 = À33.04ꢀ (c 2.0, EtOH); 1H NMR (500 MHz, CDCl3) d 7.75
(dd, J = 2.9 Hz, J = 7.5, 2H), 7.60 (t, J = 6.3, 2H), 7.37–7.42 (m, 2H),
7.23–7.33 (m, 6H), 7.18–7.22 (m, 1H), 5.53 (d, J = 7.3 Hz, 1H), 4.66
(dd, J = 5.0 Hz, J = 12.4, 1H), 4.41 (d, J = 7.0 Hz, 2H), 4.23 (t, J = 6.9,
1H), 3.77 (s, 2H), 3.75 (s, 3H), 2.88–2.99 (m, 2H); 13C NMR
(125 MHz, CDCl3) 171.2, 155.6, 143.7, 143.6, 141.2, 138.5, 128.8,
128.5, 127.6, 127.0, 126.9, 125.0, 119.9, 67.0, 53.7, 52.5, 47.0,
27.9, 25.6 ppm; IR (film) 1686 cm-1, 1526 cmÀ1, 1211 cmÀ1; HRMS
calculated for C26H25NO4Se: 495.0949. Found: 495.0954 (M + H).
High performance liquid chromatography
The HPLC analysis was carried out on a Shimadzu analytical HPLC
system with LC-10 AD pumps, SPD-10A UV-VIS detector, and
SCL-10A controller using a Symmetry™ C18 5-mm column from
Waters (4.6 Â 150 mm). Aqueous and organic phases were 0.1%
TFA in water (Buffer A) and 0.1% TFA in HPLC-grade acetonitrile
(Buffer B), respectively. Beginning with 100% Buffer A, a 1.4-ml/
min gradient elution increase of 1% Buffer B/min for 55 min
was used for all peptide chromatograms. Peptides 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 con-
troller. A Waters SymmetryPrep™ 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 in-
crease of 1% Buffer B/min for 50 min was used for all preparative
chromatograms.
General procedure for the synthesis of Fmoc-Sec(X)-OH (3a-c)
Method is adapted and optimized from the procedure of
Nicolaou [21]. Fmoc-Sec(X)-OMe 6a–c (6.48 mmol) and SnMe3OH
(2.34 g, 12.96 mmol, 2 eq) was dissolved in a 250-ml round-
bottom flask in 40-ml 1,2-dichloroethane. The reaction vessel
was purged with N2, fitted with a reflux condenser, and heated
at reflux for 1 h. At the end of this time, TLC showed consumption
of all starting material, and the reaction mixture was concen-
trated in vacuo and purified directly, without workup over silica
gel (20–40% EtOAc/Hex) to yield the final carboxylate com-
pounds 3a–c.
Fmoc-Sec(Mob)-OH (3a): (3.21 g; 97%) isolated as a colorless
amorphous solid [14].
Mass spectrometry
Fmoc-Sec(Meb)-OH (3b): (3.05 g; 95%) isolated as a colorless
amorphous solid: mp 143–145 ꢀC; [a]2D8 = À21.89ꢀ (c 2.0, EtOH);
1H NMR (500 MHz, d6 acetone) d 7.92 (d, J = 7.5 Hz, 2H), 7.81 (t,
J = 5.5 Hz, 2H), 7.48 (t, J = 7.3 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 7.16
(d, J = 7.8 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 4.58–4.68 (m, 1H),
4.37–4.50 (m, 2H), 4.33 (t, J = 7.0 Hz, 1H), 3.95 (s, 2H), 3.06–3.15
(m, 1H), 2.95–3.03 (m, 1H), 2.34 (s, 3H); 13C NMR (125 MHz, d6
acetone) 172.2, 156.5, 144.7, 144.6, 141.7, 136.8, 136.6, 129.5,
129.4, 128.2, 127.6, 125.8, 120.4, 67.0, 54.8, 47.6, 30.3, 27.4, 25.4,
20.7 ppm; IR (film) 1684 cmÀ1, 1533 cmÀ1, 736 cmÀ1; HRMS calcu-
lated for C26H25NO4Se: 495.0951. Found: 495.0956 (M + H).
Fmoc-Sec(Bzl)-OH (3c): (3.09 g; 99%) isolated as a light yellow
amorphous solid: mp 74–75 ꢀC; [a]2D8 = À12.75ꢀ (c = 2.0, EtOH);
1H NMR (500 MHz, CDCl3) d 11.48 (s, 1H), 7.69, (d, J = 7.4 Hz, 2H),
7.55 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.25 (t, J = 7.4 Hz,
2H), 7.18–7.23 (m, 3H), 7.12–7.17 (m, 2H), 5.62 (d, J = 4.0 Hz, 1H),
The MALDI-TOF mass spectrometry 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/ACN buffered to 0.1% TFA.
DTNP deprotection assay conditions for VTGGU(X)A test peptides 7–9
One-milligram (~1.7mmol) aliquots of VTGGU(X)A test peptides
were dissolved in 200 ml of either 100% TFA or 2% thioanisole/
TFA to a final concentration of ~8.5 mM. Each of these solutions
was incubated with different concentrations of DTNP 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
using centrifugation. Following drying of the pellets, the crude
isolates were dissolved in 750 ml of 0.37 M aq. NaBH4 for 20 min. At
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 155–162

DTNP FOR SELENOCYSTEINE DEPROTECTION
the end of this time, the mixture was brought to 0 ꢀC and excess
borohydride was destroyed with a few drops of AcOH. The solution
was directly subjected to analytical HPLC analysis.
was isolated using centrifugation. Following drying of the
pellets, the crude isolates were dissolved in 2-ml 0.37 M aq.
NaBH4 for 20 min. At the end of this time, the mixture was
brought to 0 ꢀC, and excess borohydride was destroyed with
a few drops of AcOH. The solution was allowed to remain in
contact with air for 30 min and then subjected to analytical
HPLC analysis [m/z: 1104.1 (M + H)].
DTNP deprotection assay conditions for protected Sec-oxytocin test
peptides 10–12
One-milligram (~0.8 mmol) aliquots of each protected Sec-
oxytocin peptide were dissolved in 200ml of either 100% TFA or
2% thioanisole/TFA to a final concentration of ~3.8mM. Each of
these solutions was incubated with different concentrations of
DTNP with agitation with time and temperature parameters as
reported in Table 1. At the end of the reaction time, cold diethyl
ether was added to each reaction and the crude precipitated
product was isolated using centrifugation. Following drying of the
pellets, the crude isolates were dissolved in 0.75-ml 9 : 1 H2O/ACN
and the resulting solutions were evaluated using analytical HPLC.
Results and Discussion
This research endeavor was composed of two distinct parts: (1) a
synthetic component involving the construction of the Sec(Meb)
and Sec(Bzl) Fmoc derivatives required to evaluate the effective-
ness of this deprotection methodology and (2) an evaluation
component in which peptide models bearing these protected
Sec derivatives were assayed as to the effectiveness of their
deprotection using DTNP. Because the achievement of the latter
depended upon the successful chemical syntheses of the former,
our efforts were first focused on the synthesis of the protected
Fmoc derivatives.
DTNP-mediated diselenide cyclization conditions for Mob-protected
Sec-oxytocin test peptide 10
5.0 mg (~4 mmol) of Mob-protected Sec-oxytocin peptide was dis-
solved in 1.0 ml of TFA. Two equivalents of DTNP dissolved in a
minimal amount of TFA was then added with brief rapid stirring.
The solution was incubated for 1 h, at the end of which cold
diethyl ether was added to the reaction and the crude precipi-
tated product was isolated using centrifugation. Following drying
of the pellet, the crude isolate was dissolved in 0.75 ml 9 : 1 H2O/
ACN and evaluated using analytical HPLC [m/z: 1104.1 (M + H)].
Synthesis of Se-Protected Fmoc-Sec Derivatives
Our decision to carry out the synthesis of the model peptides for
this study exclusively using Fmoc methodology necessitated the
construction of the Fmoc-Sec(Meb) and Fmoc-Sec(Bzl) deriva-
tives. We ultimately settled for two distinct synthetic schemes
(Figure 3). The first synthesis (Procedure 1) was based upon an
optimization of the procedure presented by Raines [18], in which
selenocystine 1 was reduced with NaBH4 and subsequently
Se-alkylated with the corresponding benzyl chloride to afford
intermediates 2a–c. Subsequent Fmoc-protection yielded the
completed Fmoc-Sec derivatives 3a–c. Yields of 3a–c varied
reproducibly using this method, with the benzyl-protected 3c
typically affording the best yields, followed by more modest
yields for the Meb-protected variant 3b and low yields for the
Mob-protected derivative 3a.
DTNP/NaBH4-mediated diselenide cyclization conditions for
Meb-protected and Bzl-protected Sec-oxytocin test peptides 11–12
Of Meb-protected and Bzl-protected Sec-oxytocin peptides,
5.0 mg (~4 mmol) each were dissolved in 1.0 ml of 2%
thioanisole/TFA. Each of these solutions was incubated with
20 eq DTNP with agitation for 1 h at 50 ꢀC followed by 4 h at
25 ꢀC. At the end of the reaction time, cold diethyl ether was
added to the reaction and the crude precipitated product
Table 1. DTNP deprotection parameters and results on model Sec-containing peptide
Protecting
group P
Peptide
synthetic
yield
Mass (m/z)
M + H
Deprotection conditions
Equivalent
(mM)
Reaction time
% deprotection/cyclization
(measured using HPLC)
(h)/temperature (ꢀC)
DTNP/TFA
Mob (7)
Meb (8)
Bzl (9)
46
72
71
675.9
659.8
646.0
0.38 (3.5)
3.3 (30)
11 (100) a
1/25
1/25
1/25
100
93
90
Mob (10)
Meb (11)
Bzl (12)
43
17
16
1346.0
1314.3
1286.0
2 (18)
1/25
1/50
1/50
98b
95c
95c
20 (183)a
20 (183)a
a2% Thioanisole added.
bYield of spontaneously cyclized peptide.
cYield of two-step induced cyclization from bis-Npys intermediate.
J. Pept. Sci. 2012; 18: 155–162 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

SCHROLL, HONDAL AND FLEMER
Although the Fmoc-Sec(Mob) derivative 3a is commercially
available, its cost was a deterring factor for us, so we decided
to seek another synthetic approach that would provide us the
considerable amounts of the Mob-protected derivative that we
would need at a much lower overall cost. We ultimately settled
for an optimized combination of two synthetic approaches based
upon the syntheses presented by Dawson [19] and van der Donk
[20]. In this synthetic design, Fmoc-protected tosylate 4 became
an advanced intermediate onto which selenium delivery was
achieved via OT displacement of an in situ-generated benzyl-
templated selenol functionality. Diselenides 5a–c were synthe-
sized according to the literature procedure [20] and used to
deliver the protected selenium to intermediate 4 upon reduction
with borohydride. Because of the lability of the Fmoc group
towards basic conditions, we were concerned that the use of
borohydride would lead to decomposition of our starting material
4. However, diselenide reduction using two equivalents of
borohydride while maintaining the reaction at 0 ꢀC allowed clean
conversion to the desired benzyl-templated Sec methyl esters
6a–c without observed Fmoc degradation. Although we were again
concerned about the integrity of the Fmoc group in the hydrolysis of
methyl esters 6a–c to their corresponding carboxylates 3a–c, we
found that treatment of these intermediates with unusual and effec-
tive reagent SnMe3OH in refluxing 1,2-dichloroethane [21] allowed
for extremely facile transformation to carboxylates 3a–c.
of these Sec-containing peptides was constructed to illustrate a
different facet of the scope of this deprotection technique. We
wished to utilize a peptide model that would compare the
robustness of the three different protecting groups towards
deprotection under the DTNP conditions. It was also of interest
to utilize an additional peptide model that would demonstrate
a merging of this basic deprotective process with concomitant
diselenide formation.
As shown in Table 1, two model peptides were employed to
illustrate the effectiveness of this deprotection methodology
and its amenability towards further synthetic manipulation. Sec-
containing hexamer test peptides 7–9 were chosen because of
their use as successful models in the analogous prior publication
by us [17] and were found to illustrate nicely the Se-deblocking
proficiency of the DTNP conditions against the three protecting
groups under study. Oxytocin analogs 10–12 were selected as
models for concomitant diselenide formation subsequent to
DTNP deprotection to further illustrate the practical use for the
process. Syntheses of hexamer test peptides 7–9 proceeded
cleanly and without difficulty. However, some difficulty was
encountered in the syntheses of oxytocin analogs 10–12.
Although construction of the Sec(Mob) oxytocin 10 proceeded
smoothly, Sec(Meb)- and Sec(Bzl) analogs 11 and 12 encoun-
tered problems with only partial attachment of one of the Sec
residues as shown using crude HPLC of the product peptide (data
not shown). Preparative HPLC allowed for clean excision of the
desired peptide from the product mixture, albeit in diminished
yield. Nevertheless, the recovered amounts of Sec(Meb)-oxytocin
and Sec(Bzl)-oxytocin were sufficient for the DTNP deprotection
assays to follow.
There was a marked difference in yields of intermediates 6a–c
dependent upon the medium of delivered borohydride. We
noticed that the use of NaBH4 in a partially aqueous medium,
although giving reasonable yield for Sec(Bzl) intermediate 6c,
afforded moderate to poor yields of Sec(Mob) and Sec(Meb)
intermediates 6a and 6b. The use of Super Hydride™ in THF gave
much more satisfactory yields of intermediates 6a–c.
Deprotection/Cyclization of Sec-Containing Model Peptides
Probing the general deprotective abilities of this methodology
made use of hexamer peptide systems 7–9. The general protocol
of the deprotection assay involved incubation of the model pep-
tide with varying concentrations of DTNP in TFA either in the
Synthesis of Sec-Containing Model Peptides
With the protected Sec derivatives in hand, we then proceeded
to use them in the syntheses of two model peptide systems. Each
Figure 4. Results of DTNP assay on protected Sec-containing peptides 7–9.
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DTNP FOR SELENOCYSTEINE DEPROTECTION
presence or absence of thioanisole. Control reactions were car-
ried out via incubation of the test peptides in 2% thioanisole/
TFA without added DTNP. Following isolation of the crude
peptide pellet via Et2O trituration and centrifugation, treat-
ment of the crude isolate with excess NaBH4 in water was
carried out to homogenize the complicated deprotection mix-
ture. Following quenching of the NaBH4, the free selenols
quickly dimerized and were quantified as their corresponding
diselenides via HPLC assay. As shown in Figure 4, the results
of these general assays illustrate some interesting differences
and parallels when compared with their corresponding Cys-
protected analogs [17].
As reported elsewhere [16], the Sec(Mob) deprotection was
effected using only stoichiometric amounts of DTNP in the
absence of thioanisole, illustrating the effectiveness of this meth-
odology towards what has traditionally been a very robust Sec
protecting group. Sec(Meb) deprotection, although admittedly
requiring somewhat higher DTNP concentration, was brought
about in a comparatively facile fashion. It was found that the
addition of thioanisole to the reaction mixture effected a some-
what superior deprotection, but similar to Se-Mob deprotection,
ultimately full Se-Meb deprotection was achievable at low DTNP
concentrations whether thioanisole was present or not. The Sec
(Bzl) deprotection offered the most promising avenue of ortho-
gonality to the process. It was found that Se-Bzl was quite robust
to the deprotection conditions, even at very high concentrations
of DTNP (<10% Bzl deprotection) in the absence of thioanisole,
whereas it proved quite labile if 2% thioanisole was added to
the reaction milieu (>90% deprotection). This ‘orthogonality hole’
in response to the different reaction conditions has potential to
provide a very practical tool for peptide syntheses requiring
post-synthetic stepwise preparation of multiple diselenide-
containing peptides.
Oxytocin-based peptides 10–12 were employed to illustrate
any propensity of this deprotective process to mediate con-
comitant diselenide formation following deprotection as part
of a desired ‘one-pot’ procedure. Following some optimization,
we were delighted to find that Mob-protected oxytocin system
10 underwent deprotection and spontaneous cyclization to the
diselenide using 2 eq DTNP in the absence of thioanisole
(Figure 5). This deprotection/cyclization sequence afforded a
very pure product as shown in the HPLC trace in Figure 5. This
result further marked the first time concomitant cyclization
accompanied a deprotection using these conditions and stands
in stark contrast to the behavior of the analogous Cys(Mob)-
containing oxytocin analogs investigated previously [17], in
which a (bis)-Npys intermediate instead formed and required
further manipulation to induce cyclization to its corresponding
disulfide.
In contrast to the ease with which Se-protection was removed in
peptides 7–9, (Meb)-protected and (Bzl)-protected oxytocins 11
and 12 required more forcing conditions to effect complete depro-
tection of both Sec residues. Indeed, 20 eq DTNP in 2% thioanisole/
TFA at a temperature of 50 ꢀC was required to fully deprotect both
of these model systems. Moreover, the consistent isolated reaction
product was not the cyclized product derived from 10 but rather
the (bis)-Npys intermediate as shown in Figure 5. This intermediate,
however, was converted to the desired cyclized seleno-oxytocin
albeit in a decidedly less elegant fashion using aqueous NaBH4
reduction, and following quenching of the borohydride environ-
ment, spontaneous cyclization afforded the desired diselenide
product in a remarkably singular fashion.
Figure 5. Deprotection/cyclization sequence for oxytocin peptides 10–12, graphically illustrating the different requirements between the Sec(Mob)-
bearing peptides and the Sec(Meb) or Sec(Bzl)-bearing peptides.
J. Pept. Sci. 2012; 18: 155–162 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

SCHROLL, HONDAL AND FLEMER
Conclusions
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Acknowledgements
These studies were supported by National Institutes of Health
Grant GM094172 to R. J. H. and Vermont Genetics Network Grant
P20 RR16462 to A. L. S.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 155–162