Fmoc-Sec(Xan)-OH: Synthesis and utility of Fmoc selenocysteine SPPS derivatives with acid-labile sidechain protection

Article abstract of DOI:10.1002/psc.2723

We report here the synthesis of the first selenocysteine SPPS derivatives which bear TFA-labile sidechain protecting groups. New compounds Fmoc-Sec(Xan)-OH and Fmoc-Sec(Trt)-OH are presented as useful and practical alternatives to the traditional Fmoc-Sec-OH derivatives currently available to the peptide chemist. From a bis Fmoc-protected selenocystine precursor, multiple avenues of diselenide reduction were attempted to determine the most effectivemethod for subsequent attachment of the protecting group electrophiles. Our previously reported one-pot reduction methodology was ultimately chosen as the optimal approach toward the synthesis of these novel building blocks, and both were easily obtained in high yield and purity. Fmoc-Sec (Xan)-OH was discovered to be bench-stable for extended timeframes while the corresponding Fmoc-Sec(Trt)-OH derivative appeared to detritylate slowly when not stored at -20 °C. Both Sec derivatives were incorporated into single- and multiple-Sec-containing test peptides in order to ascertain the peptides' deprotection behavior and final form upon TFA cleavage. Single-Sec-containing test peptides were always isolated as their corresponding diselenide dimers, while dual-Sec-containing peptide sequences were afforded exclusively as their intramolecular diselenides.

Full text of DOI:10.1002/psc.2723

Research Article
Received: 18 July 2014
Revised: 30 September 2014
Accepted: 13 October 2014
Published online in Wiley Online Library: 11 December 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2723
Fmoc-Sec(Xan)-OH: synthesis and utility
of Fmoc selenocysteine SPPS derivatives
with acid-labile sidechain protection
Stevenson Flemer Jr.*
ABSTRACT: We report here the synthesis of the first selenocysteine SPPS derivatives which bear TFA-labile sidechain protecting
groups. New compounds Fmoc-Sec(Xan)-OH and Fmoc-Sec(Trt)-OH are presented as useful and practical alternatives to the
traditional Fmoc-Sec-OH derivatives currently available to the peptide chemist. From a bis Fmoc-protected selenocystine precur-
sor, multiple avenues of diselenide reduction were attempted to determine the most effective method for subsequent attachment of
the protecting group electrophiles. Our previously reported one-pot reduction methodology was ultimately chosen as the optimal
approach toward the synthesis of these novel building blocks, and both were easily obtained in high yield and purity. Fmoc-Sec
(Xan)-OH was discovered to be bench-stable for extended timeframes while the corresponding Fmoc-Sec(Trt)-OH derivative appeared
to detritylate slowly when not stored at À20 °C. Both Sec derivatives were incorporated into single- and multiple-Sec-containing test
peptides in order to ascertain the peptides’ deprotection behavior and final form upon TFA cleavage. Single-Sec-containing test
peptides were always isolated as their corresponding diselenide dimers, while dual-Sec-containing peptide sequences were
afforded exclusively as their intramolecular diselenides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Keywords: selenocysteine; xanthenyl protection; sidechain protection; solid phase peptide synthesis
construction of the peptide sequence. In recent years, Boc SPPS
Introduction
has given way to the more gentle and straightforward piperidine-
driven Fmoc Nα-deprotection methodology [7] which typically uti-
Selenocysteine (Sec, U) is an uncommon proteinogenic amino acid
whose significance in biological processes and biochemical struc-
ture is noteworthy. Considered colloquially to be the ‘21st Amino
Acid’, Sec has utility as a structural component of active sites in var-
ious redox proteins [1], cellular signaling processes [2], and acts as a
biological selenium donor when incorporated into Sec-bearing pro-
teins [3,4]. In biological systems, selenocysteine is synthesized and
ribosomally inserted into proteins through an expanded transla-
tional process involving the conversion of free serine into Sec and
its subsequent co-translational insertion into the linear sequence
of the protein by way of a Selenocysteine Insertion Sequence
(SECIS) element [5]. There are a growing number of known SeC-
containing proteins such as selenoprotein P [4] and selenoprotein
W [6] which utilize the unusual chemistry inherent to Sec in order
to carry out diverse biochemical processes crucial to the smooth
operation of biological systems.
As interest in the field of selenoproteins grows, the demand for
analytical research samples of these biomolecules increases in kind.
The growing importance of biologically relevant Sec-containing
peptides, particularly those which may contain non-proteinogenic
amino acids or unnatural architecture, creates a niche in which
these peptides can be more easily constructed through chemical
synthesis rather than biological vectors. Solid phase peptide
synthesis (SPPS) allows for the straightforward synthesis of
Sec-containing peptides through the use of Boc or Fmoc SPPS
building blocks in which the reactive selenol sidechains of the
selenocysteine residues are protected throughout the linear
lizes orthogonal, acid-labile protecting groups on the reactive
sidechains of the amino acid derivatives. At present, all commer-
cially available Fmoc derivatives of the standard amino acids bear-
ing potentially reactive sidechains are orthogonally protected with
an acid-labile vector, mostly consisting of Trt/tBu architecture. Thus,
treatment of the peptide resin with trifluoroacetic acid (TFA) at the
completion of the linear sequence serves the dual purpose of
liberating the completed peptide from the solid support as well
as concurrent global deprotection of all sidechain protectants.
Selenocysteine is the only SPPS derivative which does not possess
TFA-labile sidechain deprotection.
Traditional Sec SPPS derivatives, whether of the Fmoc or Boc va-
riety, have exclusively employed benzyl-type sidechain protection
architecture [8–11] which (dependent upon the identity and de-
gree of substitution pattern upon it) require a variety of harsh treat-
ment conditions to effect their removal (Figure 1). More current Sec
sidechain protection protocol has recently been reported which of-
fers structural diversity and expanded orthognality when compared
with these benzyl protectants in use for decades [12]. However,
*
Correspondence to: Stevenson Flemer Jr., Department of Biochemistry, Room B415
Given Bldg, 89 Beaumont Ave, University of Vermont, Burlington, VT 05405, United
States. E-mail: sflemer@uvm.edu
Department of Biochemistry, University of Vermont, Room B415 Given Bldg, 89
Beaumont Ave, Burlington, VT 05405, United States
J. Pept. Sci. 2015; 21: 53–59
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

FLEMER
Figure 1. Traditional sidechain protection architecture for Sec SPPS derivatives.
these blocking groups also require further treatment with other re-
agents in addition to standard TFA deprotection in order to fully re-
move them. Further, these additional steps can involve harsh or
overly toxic reagents, potentially altering the peptide product pro-
file. Thus, it would be very advantageous for peptide chemists using
selenocysteine to have access to an Fmoc Sec SPPS derivative in
which the selenol sidechain could be completely and cleanly re-
moved using a standard TFA/scavenger deprotection cocktail in
analogous fashion to all other standard acid-labile sidechain
protectants.
Presented here are the results of a research effort which offers a
practical solution to this problem, allowing a more streamlined ap-
proach toward the synthesis of Sec-containing peptides. The TFA-
labile Xanthenyl (Xan) and Trityl (Trt) protecting groups were se-
lected as candidates for this study (Figure 2) since they have both
found use as sidechain protectants for their analogous cysteine
SPPS residues [13,14]. The building block derivatives Fmoc-Sec(-
Xan)-OH 2 and Fmoc-Sec(Trt)-OH 3 were easily synthesized from
bis-Fmoc selenocystine intermediate 1 as reported previously
[15,16], and incorporated into a number of test peptide sequences
to ascertain their potential and applicability toward Fmoc SPPS.
Ease of deprotection using a standard TFA cocktail was studied in
these test peptides, with careful observation toward product profile
optimization during acidolytic cleavage.
7-azabenzotriazole (HOAt) were purchased from RS Synthesis
(Louisville, KY). Piperidine and N,N-disopropylcarbodiimide were
purchased from Sigma-Aldrich (Milwaukee, WI). Novasyn TGR resin
and Fmoc-OSu were purchased from Novabiochem (EMD
Chemicals; Philadelphia, PA). L-Selenocystine, 9-hydroxyxanthene,
trifluoroacetic acid, and all other solvents and reagents were pur-
chased from Fisher Scientific (Pittsburgh, PA).
High Performance Liquid Chromatography (HPLC)
HPLC analysis was carried out on a Hitachi analytical HPLC system
with L-7100 pumps, an L-7200 autosampler, an L-7400 UV detector,
and an L-7000 interface using a Poroshell 120 column from
Agilent (4.6× 150 mm). Aqueous and organic phases were
99:1 water/acetonitrile (0.1% TFA) (Buffer A) and 90:10
acetonitrile/water (0.1% TFA) (Buffer B), respectively. Beginning
with 100% Buffer A, a 1.0ml/min gradient elution increase of 1%
Buffer B/min for 55min was used for all peptide chromatograms.
Peptides were detected at 214 nm.
Mass Spectrometry
Samples were analyzed using an AB Sciex 4000 QTrap (AB Sciex,
Framingham, MA) hybrid triple quadrupole/linear ion trap liquid
chromatograph-mass spectrometer (LCMS). Positive electrospray
ionization (ESI) was used as the ionization source. Source tempera-
ture was maintained at 400 °C. The mass spectrometer was oper-
ated in single quadrupole mode, scanning from 200 to 1000 da.
Materials and Methods
High Resolution Mass Spectrometry (HRMS)
Materials
HRMS spectra were obtained using a Thermo-Fisher Scientific LTQ
Linear Quadrupole Ion Trap-Oribitrap Mass Spectrometer Plus Liq-
uid Chromatography.
All standard Fmoc amino acid derivatives, O-benzotriazole-N,N,N′,N
′-tetramethyluronium hexafluorophosphate (HBTU), and 1-hydroxy-
Figure 2. bis-Fmoc selenocystine synthetic intermediate 1 and Fmoc-Sec(Xan)-OH 2 and Fmoc-Sec(Trt)-OH 3 derivatives presented in this study.
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ACID-LABILE SEC SIDECHAIN PROTECTION
Nuclear Magnetic Resonance (NMR)
Fmoc-Sec(Trt)-OH was isolated as a light yellow amorphous solid
which required immediate storage at À20 °C (705 mg, 87%). mp
150–152 °C; [α]2D5 = +3.75° (c = 100 mm, acetone); 1H NMR
(500 MHz, DMSO d6) δ 12.73 (br. s, 1H), 2.89 (d, J = 7.5 Hz, 2H),
7.80 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.43–7.38 (m, 2H),
7.36–7.20 (m, 17H), 4.35–4.20 (m, 3H), 4.02–3.95 (m, 1H), 2.78
(t, J = 10.5 Hz, 1H), 2.48 (dd, J = 5.0 Hz, J = 11.5 Hz, 1H); 13C
NMR (125MHz, DMSO d6) δ 172.0, 155.7, 147.7, 145.0, 143.7,
143.6, 140.6, 129.4, 128.0, 127.7, 127.6, 127.4, 127.0, 126.7, 126.5,
1H and 13C NMR spectra were obtained on a Bruker AXR 500-MHz
high-field NMR spectrometer fitted with a direct detection liquid
probe using standard pulse sequences. Spectra were obtained in
d6 DMSO, and chemical shifts are given in ppm against the center
DMSO multiplet peak at 2.51 ppm. Synthesis of (Fmoc-Sec-OH)2
(1), Procedure and characterization as previously described [16].
Synthesis of Fmoc-Sec(Xan)-OH (2): 0.5-g (0.64 mmol) bis-Fmoc-L-
selenocystine (1) and 820-mg (20 eq) finely powdered Zn were
suspended in 10-ml diethyl ether followed by the addition of 4-ml
3м HCl using our own modification [16] from the procedure of Santi
[17]. This mixture was allowed to stir vigorously for 45 min until the
organic layer turned from light yellow to colorless. At the end of this
time, the organic layer was drawn off and passed through a pad of
granulated MgSO4 in order to remove residual water. The resultant
supernatant was introduced to a 100-ml flask, and the solvent was
removed via evaporation under a stream of nitrogen with gentle
warming of the flask. Following complete concentration, 5-ml di-
chloromethane and 95-μl (1.28 mmol) TFA was added with stirring
according to the procedure of Barany [13]. 9-Hydroxyxanthene
(267 mg (1.34 mmol)) was added to this stirring solution as a solid
in small portions over 10 min, and the mixture was allowed to stir
for 1 h. At the end of this time, much of the product had precipi-
tated out of solution. Fifty-milliliter petroleum ether was added,
and the mixture was stirred vigorously for an additional 15 min at
0 °C. The resultant solid was then filtered, washing with 25ml of
ice-cold 3:1 petroleum ether/dichloromethane to yield pure
Fmoc-Sec(Xan)-OH as an off-white solid with a bluish tinge
(614 mg, 84%). mp 158–160 °C; [α]2D5 = À5.93° (c = 100 mm, ace-
125.2, 120.0, 65.6, 64.1, 53.7, 46.5, 28.2ppm; IR (film) 1722 cmÀ1
,
1034 cmÀ1, 737cmÀ1; HRMS Calcd. for C37H31NO4SeNa: 656.1311.
Found: 656.1305 (M+ Na).
Peptide Synthesis
All peptides were synthesized manually via Fmoc protocol on a 40-
μmol scale using Novasyn™ TGR resin (0.29 mmol/g). All Sec amino
acid derivatives were coupled using a fivefold molar excess of Fmoc
amino acid, DIC, and HOAt (with 5-min preactivation) in DMF for
2 h. All other amino acid couplings were carried out using a five mo-
lar excess of Fmoc amino acid, HBTU, and HOAt (with 5 min
preactivation) in 0.2 м DIEA/DMF for 1 h. Fmoc deprotection was
achieved using 20% piperidine/DMF (2× 5 min). Cleavage of pep-
tides from their resins was accomplished through treatment of
the resin with 96:2:2 TFA/TIPS/H2O for 1.5h. Following isolation,
the TFA supernatant was evaporated to one tenth its original vol-
ume under a stream of nitrogen followed by precipitation of the
crude peptide into cold anhydrous diethyl ether.
1
tone); H NMR (500MHz, DMSO d6) δ 12.86 (br. s, 1H), 7.88 (d,
Results and Discussion
J= 7.5Hz, 2H), 7.77 (d, J = 8.0Hz, 1H), 7.72 (dd, J= 3.0 Hz, J = 7.5Hz,
2H), 7.46 (dd, J = 1.5Hz, J =7.5 Hz, 1H), 7.44–7.37 (m, 3H), 7.33–
7.26 (m, 4H), 7.17–7.09 (m, 4H), 5.82 (s, 1H), 4.35–4.17 (m, 3H),
4.13–4.05 (m, 1H), 2.90 (dd, J = 5.0Hz, J = 12.5Hz, 1H), 2.80 (dd,
J= 10.0 Hz, J = 12.5Hz, 1H); 13C NMR (125 MHz, DMSO d6) δ 172.2,
155.8, 151.65, 151.62, 143.66, 143.65, 140.6, 128.7, 128.6, 128.4,
127.5, 127.0, 125.18, 125.16, 123.6, 123.5, 123.11, 123.09, 120.0,
A focus of this project was to optimize the synthetic pathway which
would afford the desired Sec derivative in the most straightforward
and facile manner. Literature preparations of Fmoc-Sec-OH deriva-
tives have traditionally followed two general reaction pathways,
based upon whether the in-situ-derived selenium nucleophile is de-
livered within the amino acid framework [10] or the protectant
module [9,18] (Figure 3). Based upon prior successful results from
our laboratories [16,18], we opted for the former approach, in which
reduction of bis-Fmoc-protected selenocystine 1 [15,16] provides a
nucleophilic selenium species which we envisioned would con-
dense with the appropriate protectant electrophile to afford the de-
sired target molecules in a one-pot process. However, since the
electrophiles in these cases are traditionally manifested as second-
ary and tertiary carbocations [13,14] as opposed to the primary ben-
zyl electrophiles commonly used for used for Sec sidechain
protection [8–12], there was some uncertainty as to whether the
in-situ-reduced Fmoc-Sec(SeH) intermediate would successfully at-
tack them. As such, we elected to carry out a survey of different
116.2, 116.1, 65.7, 54.3, 46.5, 33.9, 25.5 ppm; IR (film) 1721 cmÀ1
,
1260 cmÀ1, 760cmÀ1; HRMS Calcd. for C31H25NO5SeNa: 594.0790.
Found: 594.0783 (M + Na).; Synthesis of Fmoc-Sec(Trt)-OH (3):
Diselenide reduction of bis Fmoc Selenocystine 1 was carried out
identically as for 2. To the reduced selenol residue was added
5-ml dichloromethane followed by 373mg (1.34 mmol) trityl
chloride in small solid portions over 10min (with no TFA activator).
After 1.5h, some solid was observed to have precipitated out of
solution. At this time, 50-ml petroleum ether was added and the
mixture was stirred vigorously for an additional 15 min at 0 °C.
The resultant solid was then filtered, washing with 25 ml of
ice-cold 3:1 petroleum ether/dichloromethane to yield pure
Figure 3. Traditional literature synthetic approaches toward Sec SPPS derivatives.
J. Pept. Sci. 2015; 21: 53–59
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FLEMER
reduction conditions with concurrent electrophile attachment
based upon analogous literature preparations.
through the treatment of bis Fmoc selenocystine 1 with In(I) iodide
to afford (following oxidative insertion) the corresponding indium
(III)-dichalcogenate (Entry 2, Table 1). Ranu et al. showed that in-
situ-generated indium(III)-phenylseleno dichalcogenates con-
densed smoothly with a variety of alkyl halides to form a series of
diorganyl selenides [24]. Further, Braga illustrated that formation
and delivery of the corresponding alkylseleno derivative onto a cor-
responding electrophile were within the scope of the reaction [25].
In our hands, reaction of 1 with In(I)I appeared to circumvent the
undesired reformation of starting material 1 (as evidenced by TLC
analysis). However, treatment of the intermediate mixture with trityl
chloride resulted in no formation of 3 whatsoever. Indeed, the TLC
profile maintained constant even after prolonged reaction time,
suggesting that the putative In(III)I diorganylseleno intermediate
was not sufficiently reactive to induce its condensation with a ter-
tiary alkyl electrophile.
The Zn-mediated reductive methodology of Barany in his syn-
thesis of the analogous Fmoc-Cys(Xan)-OH derivative [13] was an
attractive approach to utilize for the synthesis of the correspond-
ing Sec(Xan) analog 2, particularly because the acidic conditions
required for the in-situ generation of the 9-Xanthenyl cation elec-
trophile doubled as an environment in which diselenide refor-
mation was sufficiently suppressed. Unfortunately, our initial
efforts using Barany’s Zn/AcOH diselenide reduction conditions
resulted in no product formation. However, aligning with the
successes we have enjoyed previously [16], adapting the reduc-
tion environment to the biphasic Zn-mediated reductive system
of Santi [17] was gratifyingly successful for Fmoc-Sec(Xan)-OH 2
(Entry 3a, Table 1). The analogous Fmoc-Sec(Trt)-OH derivative
3 was similarly obtained using the corresponding trityl chloride
electrophile (Entry 3b, Table 1). Using this approach with our
own modifications, both derivatives were isolated in yields ex-
ceeding 80%.
Ordinarily, matching reaction conditions to the analogous sulfur-
containing systems (ie: cysteine) would appear to be a reasonable
approach towards designing a comparable Sec synthetic method-
ology due to the very similar chemistry shared between the two
chalcogen elements. In contrast to sulfur, however, the high oxida-
tive potential of selenium and the propensity of a generated Sec
selenol to quickly reestablish its diselenide form [19,20] became
the principal synthetic challenge to overcome. Care had to be taken
to choose reaction conditions which allowed the reactive selenol to
remain intact long enough to allow condensation with the appro-
priate protectant electrophiles. Subsequently, a survey of diselenide
scission methodologies was undertaken in order to identify which
would be most amenable to the current structural goal (Table 1).
One of the most common approaches of selenocystine reduction
prior to Se derivatization is through aqueous borohydride reduc-
tion [10]. Our initial attempts using this methodology focused on
our previously reported anhydrous reduction conditions using Su-
per Hydride (LiBEt3H) in THF [18] to generate the reactive selenium
species (entry 1, Table 1). However, incubation of the generated in-
termediate with trityl chloride [14,21] resulted in only a trace of
Fmoc-Sec(Trt)-OH formation and nearly complete regeneration of
the original bis Fmoc selenocystine 1 regardless of how stringent
our efforts to exclude oxygen from the reaction vessel. The reason-
ing for this was undoubtedly the aforementioned propensity of free
selenol to readily form its corresponding diselenide under alkaline
conditions. This result led us to elect not to pursue other known
diselenide reduction vectors which employ alkaline conditions such
as phosphine [22] and hydrazine hydrate-alkali [23] and instead fo-
cus on methodologies which utilize non-basic conditions.
An interesting and promising approach toward diselenide reduc-
tion without the potential for impeding diselenide reformation was
Table 1. Survey of attempted synthetic routes into Sec derivatives 2 and 3
Entry
1
Reducing conditions
LiBEt3H (ref. 17)
Sec intermediate
Electrophile
Notes
Trace product formed. Selenol intermediate readily
re-formed 1 under the basic reaction conditions.
2
In(I)I (refs. 21 and 22)
Zn/HCl (refs. 15 and 16)
Zn/HCl (refs. 15 and 16)
No product formed. In(III)I diorganyl selenoate unreactive
to the tertiary alkyl chloride electrophile.
3a
3b
84% Fmoc-Sec(Xan)-OH product isolated.
87% Fmoc-Sec(Trt)-OH product isolated.
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ACID-LABILE SEC SIDECHAIN PROTECTION
It was noticed that, upon languishing on the lab bench for more
than 1 h, Sec(Trt) derivative 3 began to take on an increasingly yel-
low tinge, signifying a slow rate of spontaneous detritylation. How-
ever, it was found that if derivative 3 were stored at À20 °C, it
retained its original light yellow color indefinitely. Sec(Xan) deriva-
tive 2 on the other hand was found to be remarkably bench-stable
for extended periods (>1 month), retaining its diagnostic bluish
tinge without evidence of spontaneous decomposition. Since shelf
stability is an important consideration when weighing the merits
and practical viability of new compounds such as these derivatives,
it was decided that Fmoc-Sec(Xan)-OH 2 was the superior product,
given that both derivatives produced comparable results when in-
corporated into peptide models (vide infra).
With Fmoc-Sec(Xan)-OH 2 and Fmoc-Sec(Trt)-OH 3 derivatives in
hand, their incorporation into suitable peptide models became the
next objective of this undertaking. We wanted to select peptide
templates which would showcase the efficiency of TFA-mediated
deprotection of Sec(Xan) and Sec(Trt) as well as to explore the fate
of the liberated Sec selenol once the deprotected peptide was iso-
lated. Importantly, we wished to design peptide sequences to help
identify a niche for these TFA-labile protectants which would assist
in solving enduring practical problems and challenges in the arena
of Sec SPPS. For example, a major concern in the Fmoc synthesis of
Sec-containing peptides is the alleged propensity of protected
selenocysteine to deselenate during base-mediated peptide elon-
gation and/or cleavage to form dehydroalanine (dHA) [26]. More
generally, it was important to show that any peptides synthesized
using these derivatives retained stereointegrity at the Sec residue.
The Cys-containing Stromelysin 1 matrix metalloproteinase in-
hibitor (MMP3-I) [27] of the sequence Ac-Arg-Cys-Gly-Val-Pro-
Asp-NH2 was chosen as a general template in which to build
the Sec-containing analog in order to demonstrate ease of con-
struction of this template using Sec derivatives 2 and 3. While
incorporation of 2 and 3 into their respective MMP3-1 se-
quences was carried out under neutral DIC/HOAt conditions, all
remaining residues were coupled in a standard fashion under
basic conditions (0.2 м DIEA/DMF) using HBTU/HOAt conditions.
Additionally, no efforts were made to shorten Fmoc deprotection
sequences from the standard length of time used for all other
residues (2× 5 min). Liberation and deprotection of the peptides
from the resin were carried out using a standard 96:2:2 v/v
TFA/TIS/H2O cocktail for 1.5h. The crude HPLC traces of both
batches of the MMP3-I sequence showed a major peak compris-
ing ~90% of the profile (Figure 4). Mass spectrometric (MS) anal-
ysis of this component showed it to be the MMP3-I diselenide
dimer, presumably formed spontaneously upon cleavage of the
peptide from the resin. MS analysis of the preceding neighbor-
ing peaks present in both chromatograms showed the existence
of small amounts of enduring side products. Mass analysis of
these peaks shows evidence of two putative additional compo-
nents, dHA adduct 5 and Se-OAc conjugate 6. Since these peaks
are in the extreme minority in the overall HPLC profile, it can be
surmised that any alternate reaction pathways encountered dur-
ing synthesis and/or cleavage of this peptide were gratifyingly
minimal. It should be further noted that the analogous Cys-
containing MMP3-I sequence was similarly constructed, produc-
ing an HPLC profile which showed a similar high level of crude
peptide purity as the Sec-containing version, but manifesting
predictably as the free thiol rather than the disulfide dimer (data
not shown).
In order to offer the greatest demonstration of the abilities of de-
rivatives 2 and 3 toward the successful synthesis of Sec-containing
peptides without racemization or deselenation, we undertook the
synthesis of Moroder’s Sec-containing glutaredoxin analog frag-
ment Lys16 Grx 10–17 of the sequence Ac-Gly-Sec-Pro-Tyr-Sec-Val-
Lys-Ala-NH2 [26], wherein both Sec residues were sidechain-
protected with either Xan or Trt (Figure 5). This sequence was espe-
cially significant in that it was the model which was shown to be es-
pecially prone to deselenation under standard basic coupling and
Fmoc deprotection conditions. The same traditional synthetic con-
ditions as with the previously characterized MMP3-I analog were
carried out in this synthesis, utilizing identical cleavage protocol.
To our surprise, the resultant crude isolate was extremely homoge-
nous, affording a single peak in the HPLC profile of the crude isolate
(see chromatograms, Figure 5). MS analysis of this peak showed a
mass consistent with the intramolecular diselenide, derived
Figure 4. Chromatograms showing MS Peak analysis of crude Mmp3-I peptide isolates.
J. Pept. Sci. 2015; 21: 53–59
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FLEMER
Figure 5. HPLC chromatograms derived from crude Lys16 Grx 10–17 peptide isolates.
presumably from the spontaneous cyclization during cleavage.
Again, it is noteworthy that the bis-Cys-containing analog of Lys16
Grx 10–17 was isolated as the reduced bis-thiol compound, and
was able to be coaxed into its intramolecularly cyclized disulfide
form by 3-h incubation in mild alkaline buffer (data not shown).
The successful results from the incorporation of derivatives 2 and
3 into the two test peptide sequences were in sharp contrast to pre-
vious literature-based outcomes concerning the use of Fmoc-based
Sec derivatives in SPPS. Specifically, Moroder and coworkers re-
ported a high degree of deselenation tendency in their synthesis
of the Sec-containing Native Grx 10–17 analog [26] using Fmoc-
Sec(Mob) as the building block in the sequence. The researchers
surmised that the basic conditions of Fmoc amino acid coupling
and 20% piperidine deprotection gave rise to significant amounts
of dHA- and β-piperidyl-containing adducts derived from
deselenated Sec, requiring them to optimize the synthetic condi-
tions to use pentafluorophenyl ester amino acid building blocks
and significantly shortened piperidine deprotection sequences.
Concurrent with these augmented coupling conditions, Lys was
substituted for Arg at the 16 position in order to avoid the
challenging Arg(Pmc) deprotection in favor of the comparatively
facile Lys(Boc) removal.
It is quite noteworthy that our synthesis of the identical peptide
using Sec derivatives 2 and 3 under traditional (ie: standard basic)
conditions produced crude product profiles which possessed few
detectable side products. Indeed, the test peptide which did pro-
duce measurable side products (including the familiar dHA adduct)
was the MMP3-I model. A closer look at the identities of the puta-
tive side products in the MMP3-I model suggest a specific mecha-
nism for both of their genesis based upon partial Se oxidation
during peptide elongation (Figure 6). A selenoxide formation event
9 during peptide synthesis followed by a Pummerer-style acetyla-
tion 10 [28] would provide the impetus to form dHA adduct 5 via
base-induced elimination, driven by the ability to quench the posi-
tive charge on selenium. Alternatively, structure 10 might endure
up until the cleavage/deprotection process, releasing xanthenyl
cation 12 to afford Se-OAc conjugate 6 as the additional side prod-
uct. It is postulated that Se oxidation and any potential dHA forma-
tion would have had to occur after the installation (and subsequent
Fmoc deprotection) of the N-terminal Arg residue due to the
Figure 6. Possible mechanism to explain the existence of adducts 5 and 6 in MMP3-I test peptide product profile.
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Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015; 21: 53–59

ACID-LABILE SEC SIDECHAIN PROTECTION
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absence of β-piperidyl adducts within the crude deprotection pro-
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could be explained by the attack of intermediate 9 on Ac2O, the fi-
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might be why the similar Lys16 Grx 10–17 peptide model 7 showed
no evidence of dHA or Se-OAc adducts in its product profile, de-
spite the fact that its synthesis was carried out under identical con-
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Conclusions
From the successful synthesis of the two preceding test peptides, it
is evident that a TFA-labile vector of sidechain deprotection of Sec
is a viable and effective methodology. Two Fmoc-Sec-Oh deriva-
tives bearing TFA-labile sidechain protection, Fmoc-Sec(Xan)-OH
and Fmoc-Sec(Trt)-OH, have been synthesized and fully character-
ized. Of the two derivatives, Fmoc-Sec(Xan) was chosen as the more
viable and practical compound due to its enhanced bench stability.
Both derivatives were incorporated into test peptide systems
which, following TFA deprotection, produced diselenide peptides
in extremely high purity. Small amounts of dHA and Se-OAc
peptide side products were observed in the crude deprotection
profile of one of the peptides, and a possible mechanism was put
forth to account for their existence.
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S-trityl a key derivative to prepare N-methyl cysteines. J. Comb. Chem.
2008; 10: 69–78.
22 Sakakibara M, Katsumata K, Watanabe Y, Toru T Ueno Y. A convenient
procedure for the preparation of organic selenides. Synthesis 1992;
377–89.
Acknowledgements
I would like to acknowledge and thank Dr. Robert J. Hondal (Depart-
ment of Biochemistry; University of Vermont) for his invaluable
ideas and guidance in the writing of this manuscript.
HRMS spectra were the result of support by the Vermont Genet-
ics Network through grant number 8P20GM103449 from the INBRE
program of the National Institute of General Medical Sciences
(NIGMS), a component of the National Institutes of Health (NIH).
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of Diethyl Diselenide with Dihaloalkanes in an Alkaline Reductive
Medium. Russ J. Gen. Chem. 2003; 73: 1243–5.
24 Ranu BC, Mandal T Samanta S. Indium(I) Iodide-Mediated Cleavage of
Diphenyl Diselenide. An Efficient One-Pot Procedure for the Synthesis
of Unsymmetrical Diorganyl Selenides. Org. Lett. 2003; 5: 1439–41.
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matrix metalloproteinases inhibit human stromelysin and collagenase.
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