Preparation of the β3-homoselenocysteine derivatives Fmoc-β3hSec(PMB)-OH and Boc-β3hSec(PMB)-OH for solution and solid-phase-peptide synthesis and selenoligation

Article abstract of DOI:10.1002/hlca.200790171

The title compounds, 4 and 7, have been prepared from the corresponding α-amino acid derivative selenocystine (1) by the following sequence of steps: cleavage of the Se-Se bond with NaBH₄, p-methoxybenzyl (PMB) protection of the SeH group, Fmoc or Boc protection at the N-atom and Arndt-Eistert homologation (Schemes 1 and 2). A β³-heptapeptide 8 with an N-terminal β³-hSec(PMB) residue was synthesized on Rink amide AM resin and deprotected ('in air') to give the corresponding diselenide 9, which, in turn, was coupled with a β³-tetrapeptide thiol ester 10 by a seleno-ligation. The product β³- undecapeptide was identified as its diselenide and its mixed selenosulfide with thiophenol (Scheme 3). The differences between α- and β-Sec derivatives are discussed.

Full text of DOI:10.1002/hlca.200790171

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Preparation of the b3-Homoselenocysteine Derivatives Fmoc-b3hSec(PMB)-
OH and Boc-b3hSec(PMB)-OH for Solution and Solid-Phase-Peptide
Synthesis and Selenoligation
by Oliver Flçgel1), Giulio Casi2), Donald Hilvert, and Dieter Seebach*
Laboratorium für Organische Chemie, Department Chemie und Angewandte Biowissenschaften,
Eidgençssische Technische Hochschule, ETH-Zürich, Hçnggerberg, HCI, Wolfgang-Pauli-Strasse 10,
CH-8093 Zürich
(phone: þ 41446322990; fax: þ 41446321144; e-mail: seebach@org.chem.ethz.ch)
Dedicated, in admiration, to Professor Teruaki Mukaiyama on the occasion of his 80th birthday
The title compounds, 4 and 7, have been prepared from the corresponding a-amino acid derivative
selenocystine (1) by the following sequence of steps: cleavage of the SeÀSe bond with NaBH4, p-
methoxybenzyl (PMB) protection of the SeH group, Fmoc or Boc protection at the N-atom and Arndt –
Eistert homologation (Schemes 1 and 2). A b3-heptapeptide 8 with an N-terminal b3-hSec(PMB) residue
was synthesized on Rink amide AM resin and deprotected (ꢀin airꢁ) to give the corresponding diselenide
9, which, in turn, was coupled with a b3-tetrapeptide thiol ester 10 by a seleno-ligation. The product b3-
undecapeptide was identified as its diselenide and its mixed selenosulfide with thiophenol (Scheme 3).
The differences between a- and b-Sec derivatives are discussed.
1. Introduction. – Selenium is an essential trace element in human nutrition [1].
Recent studies have also reported beneficial effects of selenium compounds for cancer
prevention [2]. Among selenium derivatives, selenocysteine (Sec or U), the 21st
proteinogenic amino acid [3], has been the focus of considerable attention3). This
moiety is found in a variety of naturally occuring enzymes (selenoproteins) such as
glutathione peroxidase, iodothyronine deiodinase, and thioredoxin reductase [4]. The
basis for the incorporation of Sec into proteins has been established by Bçckꢁs
pioneering work on Escherichia coli mutants [5]. Selenocysteine is incorporated into
selenoproteins by a process that decodes the UGA codon as selenocysteine. For most
human selenoproteins, ꢀhomologuesꢁ exist, in which selenocysteine is replaced by
cysteine. These enzymes are normally weaker catalysts when compared to the
corresponding selenoproteins [6]. The selenol group (pKa ꢀ 5.7) is more acidic than the
thiol group (pKa ꢀ 8.5) [7]. Thus, in contrast to cysteine, selenocysteine is ionized at
physiological pH. Furthermore, the Se-atom has a higher polarizability, which makes
the selenol group a ꢀsofterꢁ SN2-type nucleophile.
1
)
Postdoctoral Fellowship at ETH-Zürich 2004/2006, financed by Deutsche Forschungsgemeinschaft,
Schweizer Nationalfonds (Project No. 200020-109065), and Novartis Pharma AG, Basel.
Part of the projected Ph.D. thesis of G.C., ETH-Zürich.
For a comprehensive review ꢀFrom Selenium to Selenoproteins: Synthesis, Identity, and Their Role in
Human Healthꢁ and for a discussion on ꢀThe Importance of Selenium to Human Healthꢁ see [2b,c].
2
3
)
)
ꢂ 2007 Verlag Helvetica Chimica Acta AG, Zürich

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Diselenides have lower reduction potential as compared to disulfides, and
selenocysteine-containing peptides form diselenides more rapidly than the correspond-
ing cysteine-containing peptides form disulfides [8]. The presence of selenium in an
amino acid sequence provides a spectroscopic as well as a mechanistic probe of peptide
properties4).
Furthermore, the selenol group of selenocysteine can be easily eliminated under
mild oxidative conditions to form dehydroalanines, which are convenient precursors for
the preparation of peptide conjugates [10]. For these reasons, interest in selenocysteine
and its derivatives has dramatically increased in recent years.
Many different syntheses of selenocysteine derivatives have been reported [11]. In
a common approach, a selenolate acts as nucleophile on an activated OH group of a
serine analog. For solid-phase peptide synthesis (SPPS) N-Boc- or N-Fmoc-protected
derivatives are the most convenient. The currently most useful protective group for a
selenol is the para-methoxybenzyl-group (PMB or Mob group) [11b,c]. The PMB
group can be removed under various conditions. For instance, Fujii and co-workers
used oxidative conditions (I2 in AcOH or DMSO in CF3COOH (TFA)) or strong
Lewis acids (Me3SiBr (TMSBr) or TMSOTf (Tf ¼ trifluoromethylsulfonyl)) [11b]. An
alternative method utilizes 2-nitrophenylsulfenyl chloride [12] in analogy to protocols
for deprotection of related cysteine derivatives [13][14]. A milder method for
deprotection of Sec(PMB) has been recently reported by Hondal and co-workers [15]:
best results were obtained using 2,2’-dithiobis(5-nitropyridine) (DTNP).
Consequently, typical building blocks for SPPS of Sec-containing peptides are
Fmoc-Sec(PMB)-OH and Boc-Sec(PMB)-OH. However, special care must be taken
while performing SPPS with these derivatives, since they are prone to racemization and
elimination under basic conditions. In particular, iterative piperidine deprotection steps
can lead to the formation of dehydroalanine moieties within the peptide and
subsequently to the corresponding piperidine adducts [16][17] (replacement of
CH2SeR by CH2ÀN(CH2)5). To minimize these problems the incorporation of
selenium residues is usually performed with preformed pentafluorophenyl esters
(OPfp esters) in the presence of 1-hydroxy-1H-benzotriazole (HOBt) [16][17], which
obviates the need for tertiary amines.
For the synthesis of larger peptides, Kentꢁs native chemical ligation, the coupling of
two unprotected peptides, has proven to be a useful tool [18]. In this convergent
synthetic strategy, a C-terminal thioester group of one peptide is coupled with an N-
terminal cysteine residue of another peptide. This technique has also been extended to
the coupling of a C-terminal thioester with an N-terminal Sec moiety5). In contrast to
the conventional native chemical (thio)ligation, where disulfide bonds are cleaved with
thiophenol, a phosphine (usually tris(2-carboxyethyl)phosphine (TCEP)) is often
added for reductive cleavage of diselenide bonds in the reaction mixture of a
(seleno)ligation [19][20]. In some cases, though, thiophenol is sufficient for this
purpose [11c][21][22]. Expressed protein ligation [23] has been used to prepare
4
)
A general overview on the replacement of sulfur by other chalcogens in peptides and proteins is
given in [9].
An overview of the use of Sec derivatives for chemoselective ligations is given in [19].
5
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Helvetica Chimica Acta – Vol. 90 (2007)
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selenoproteins [20][24]; here, the thioesters are generated by using recombinant DNA
technology and condensed with a synthetic Sec-containing segment.
b-Peptides represent a biomedically promising class of peptidomimetics. Like their
a-counterparts, b-peptides fold to secondary structures such as turns, helices, or sheets
[25]. Furthermore, they can mimic a-peptidic hormones, as well as peptide – protein
and protein – protein interactions [25a]. They may be orally bioavailable [26], and,
most importantly, they are enzymatically and metabolically stable [25a]. The b2- and b3-
amino acid homologues of the 20 proteinogenic a-amino acids have all been prepared
and incorporated in b-peptides, the properties of which have been studied extensively
[25a]. Since the 21st proteinogenic amino acid, selenocysteine (Sec), confers
interesting features to a-peptides and proteins, we decided to prepare its b3-homologue
and to explore its use in b-peptide synthesis. We here describe the preparation of Fmoc-
b3Sec(PMB)-OH and of Boc-b3Sec(PMB)-OH, as well as their use in solid phase
synthesis and in native chemical ligation.
2. Preparation of Fmoc-b3hSec(PMB)-OH and Boc-b3hSec(PMB)-OH. – Fmoc-
b3hSec(PMB)-OH (4) was synthesized starting from commercial selenocystine (1). The
SeÀSe bond of 1 was cleaved with NaBH4, and the selenol was protected with a PMB
group, according to the procedure published by Fujii and co-workers [11b], and
Vermeulen and co-workers [11e]. Subsequent treatment with N-({[(9H-fluoren-9-
yl)methoxy]carbonyl}oxy)succinimide (Fmoc-OSu) furnished Fmoc-Sec(PMB)-OH
(2) [11b] (Scheme 1) in 77% yield over three steps.
Scheme 1. Preparation of Fmoc-b3hSec(PMB)-OH (4). For abbreviations, see accompanying text.
Homologation to the b-amino-acid derivative 4 was accomplished by the Arndt –
Eistert protocol [25d][27][28], wherein the diazo ketone 3 was converted to 4 by an
ultrasound-promoted [29] Wolff rearrangement [30] with PhCOOAg.
Boc-b3hSec(PMB)-OH (7) was obtained in the same way as the Fmoc derivative
(Scheme 2). Again, the corresponding a-amino acid (a-Boc-Sec(PMB)-OH, (5)) was
first prepared, and homologation through the diazo ketone 6 led to the desired N- and
Se-protected carboxylic acid 7.
According to these simple protocols, Fmoc-b3hSec(PMB)-OH 4 and Boc-
b3hSec(PMB)-OH 7 can be prepared in five steps starting from commercial precursors
in 40 and 57% overall yield, respectively.

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Scheme 2. Preparation of Boc-b3hSec(PMB)-OH (7)
Due to the high nucleophilicity of selenium compounds of the general formula
RÀSeÀR, we had expected difficulties in the reaction with CH2N2 (2 ! 3 and 5 ! 6)
and in the Wolff-rearrangement step of the intermediate diazo ketone (3 ! 4 and 6 !
7)6)7). However, the overall yield of diazo-ketone formation and Wolff rearrangement
(2 ! 4, 5 ! 7) was comparable to that of the corresponding sulfur derivative (Fmoc-
Cys(PMB)-OH or Fmoc-Cys(Acm)-OH ! Fmoc-b3hCys(Trt)-OH) [28c]).
3. Preparation of
a b-Homoselenocysteine-Containing b-Peptide. – Boc-
b3hSec(PMB)-OH was used for the preparation of the b-peptide 8, which, in turn,
was used for a (seleno)ligation to the undecapeptide 11 (cf. Scheme 3, below).
As mentioned in the Introduction, in a-peptide synthesis, incorporation of Sec is
usually performed with the corresponding pentafluorophenyl esters and HOBt
6
)
After all, selenoethers, more so than thioethers, are expected to react with a carbene to form
selenoylides [31]: cf. the catalytic enantioselective oxirane formation by in-situ trapping of a carbene
(generated from a diazo compound) with a thioether and subsequent reaction with an aldehyde
[32].
This may suggest that no free carbene is involved in the Wolff rearrangement of 3 and 6, compatible
with the view that migration and loss of nitrogen are concerted [33][34]:
7
)

Helvetica Chimica Acta – Vol. 90 (2007)
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catalysis [16] to avoid racemization and elimination [17]. Neither of these processes is
expected to occur with b-homoselenocysteine, since the side chain is in the b- and not in
the a-position of the carbonyl group. For the synthesis of the b-heptapeptide 8 with an
N-terminal b3hSec residue, we used conventional solid-phase coupling on a Rink-
Amide AM resin. The couplings were achieved with HATU and Hünigꢁs base in DMF.
Prior to the incorporation of the b-homoselenocysteine moiety, the resin-bound b-
peptide was divided into two equal portions.
The Boc (PMB-protected) b-homoselenocysteine was coupled to the first half of
the resin-bound peptide by standard methods (HATU and DIPEA as tertiary base).
The use of N-Boc-protected b-selenocysteine obviates the need for a final, N-terminal
Fmoc deprotection. After this last coupling step, the peptide was removed from the
resin under conditions that did not set the selenol groups free. After purification by
preparative HPLC, the heptapeptide 8 was isolated as its TFA salt in a disappointingly
low 21% yield.
Alternatively, b-homoselenocysteine was attached to the other half of the resin-
bound peptide by activation as its pentafluorophenyl (PFP) ester8) in the presence of
HOBt. After cleavage from the resin and purification by preparative HPLC, the
heptapeptide 8 was isolated as its TFA salt in a similarly low 19% yield.
When samples of the heptapeptide 8 were treated with a mixture of m-cresol,
thioanisol, TFA, and TMSBr (50 :120 :750 :132) for 1 h at 08, followed by preparative
HPLC purification, the diselenide 9 was isolated as its TFA salt in 59% yield9).
Thus, both methods of attaching the b3hSec to the resin-bound b-hexapeptide
afforded the b-heptapeptide 8 (in comparable overall yield). As expected, base
treatment does not seem to have an adverse effect on the results in the case of b3hSec
derivatives.
Boc-b3hSec(PMB)-OPFP was prepared according to [35].
Additionally, 18% starting material 8 was recovered.
8
)
)
9

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4. Selenoligation. – Coupling of the b-heptapeptide fragment derivative 9 with the
b-tetrapeptide thiol ester10) 10 was achieved by Sec-mediated chemical ligation
(selenoligation [11c][20][21]) as depicted in Scheme 3.
The TFA salts of the lyophilized peptides were transferred to a Schlenk flask, and
degassed phosphate buffer (100 mm, pH 8.5) containing 6m GdmCl was added to
dissolve them. After adjusting the pH of the solution to 7.5, the reaction was initiated
with 5% thiophenol (v/v) under N2. The course of the reaction was monitored by HPLC
and LC/MS analyses. The conversion was judged to be complete after 8 h11). The
disappearance of the starting materials was accompanied by formation of two new
compounds (Fig. 1): the faster moving species corresponded to the diselenide 11c of
peptide 11a ([M þ H]þ, C136H205N26O32Se2þ ; found: 2874.0; calc. 2874.3 Da)12), whereas
the slower moving species had a mass which is compatible with the selenosulfide 11b
formed from peptide 11a and thiophenol ([M þ H]þ, C74H108N13O16SSeþ; found:
1546.8; calc. 1546.7 Da).
After completion of the reaction, the ligation mixture was treated with dithio-
threitol (DTT; 4 mg/ml reaction) for 30 min under N2 in order to generate a
homogenous solution that was subsequently washed twice with Et2O. The resulting
aqueous solution was diluted with 3 ml of MeCN/H2O 1:1 containing 0.1% of TFA, and
then directly injected into the preparative HPLC column. The diselenide product 11c
was isolated as the TFA salt in 50% yield and characterized by analytical HPLC and
high-resolution MALDI-MS (Fig. 2).
The normalized CD spectra in MeOH of the b-peptidic diselenides 9 and 11c, and of
the mixed selenosulfide 11b, are shown in Fig. 3. The starting diselenide 9 and the
selenosulfide product 11b exhibit the characteristic Cotton effect of a 314-helix: a
minimum around 214 nm and a maximum near 200 nm. The diselenide 11c of the
ligation product shows, instead, a minimum near 208 nm but no positive Cotton effect at
shorter wavelength.
5. Discussion and Conclusions. – The Arndt – Eistert-homologation sequence of
steps is readily applicable to selenocysteine (H-Sec-OH) for the preparation of Boc or
Fmoc protected b3hSec derivatives in good yields (57 and 40%, resp., for five steps from
selenocystine). Furthermore Fmoc-b3hSec(PMB)OH was successfully employed for
selenoligation of a b-hepta- and a b-tetrapeptide to give derivatives 11b and 11c of the
b-undecapeptide 11a.
As noted in the Introduction, Sec residues are convenient precursors of the
dehydroalanine moiety A, the C¼C bond of which is captodatively [37] substituted.
Analogous elimination with the b3hSec residue would lead to a dehydro-b3hAla unit B
in which the C¼C bond is substituted by an acylamido and a CH2CO group. The C¼C
bond in B is expected to be a better donor for electrophiles than that in A. The product
of electrophilic attack on B, an acyliminium ion C, is likely to lose a proton from the a-
10
)
The preparation of the thioester 10 is described in [36]. We thank Dr. Thierry Kimmerlin for the
preparation of the sample we used (cf. ETH Dissertation No. 15800, p. 83, Zürich, 2004).
For a comparison HPLC chromatograms after 0, 5 and 18 h see Fig. 1.
The masses reported in the discussion were obtained from LC/ESI-MS. The exact masses are
reported in the Exper. Part.
11
12
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Helvetica Chimica Acta – Vol. 90 (2007)
Fig. 1. Analytical HPLC traces of the ligation reaction between diselenide 9 and thioester 10. At time zero
(without thiophenol), the two starting materials are visible. Five hours after PhSH addition, the coupling
products 11b and 11c accumulated. After 18 h, the starting diselenide 9 was almost completely
consumed, and the thiophenol ester 10’ (SPh instead of S(CH2)2CO2Et in 10) as well as the ꢀmixedꢁ
selenosulfide 9’ (PhSSe derivative formed from 9) were identified by LC/ESI-MS. Chromatographic
conditions: C8 column (Macherey-Nagel 250 mm ꢁ 4.6 mm ꢁ 300 Š, 5 m; 5 – 95% B in 60 min: 5 min 5%
B, 50 min 60% B, 55 min 95% B).
carbonyl position to give an a,b-unsaturated b-acylamino-carbonyl system D (ꢀvinyl-
ogous amideꢁ). With E ¼ H in C and D, this process would be a simple double-bond
shift, and, with other electrophiles E, there would be a possibility of elaborating the
structure further.
The CD spectrum of the diselenide 11c (green curve in Fig. 3), as compared to
those of the b-peptides 9 and 11b, provides yet another example of the structural
changes that are seen when long-chain b-peptides are investigated. Based on an
analogy to many structurally characterized b-peptides that exhibit a negative Cotton
effect near 215 nm and a positive Cotton effect near 200 nm, the CD spectra shown in
Fig. 3 suggest that b-peptides 9 and 11b, which contain 14 and 11 b-amino-acid residues,

Helvetica Chimica Acta – Vol. 90 (2007)
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Fig. 2. HPLC Trace and high-resolution MALDI mass spectrum of peptide diselenide 11c. a) Analytical
HPLC chromatogram of purified 11c; conditions: C8 column (Macherey-Nagel 250 mm ꢁ 4.6 mm ꢁ
300 Š, 5 m; 5 – 95% B in 60 min: 5 min 5% B, 50 min 60% B, 55 min 95% B). b) High resolution
MALDI of 11c. Shown is the isotopic pattern of [M þ H]þ between the sixth and the seventeenth isotopic
peak. A value of 2874.365 was calculated for the twelfth isotopic peak based on a full width at half
maximum resolution of 30,000 using the Exact Mass Calculator program from the IonSpec Corporation.
The mass found for the corresponding peak was 2874.359.

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Fig. 3. Normalized CD Spectra of 9 and 11. The spectra were recorded in MeOH, at 208, with 0.25 mm 9,
0.19 mm 11c, and 0.18 mm 11b. Solutions were prepared by weighing the peptide on a mg-precision
balance and dissolving it in a volumetric flask.
respectively, adopt 314-helical structures [25]. Hypsochromic shifts of the negative and
the ꢀlossꢁ of the positive Cotton effect, as seen in the CD spectrum of the diselenide 11c,
have been observed previously with larger b-peptides [25a,b,e][36][38] but never
interpreted. As we and others have noted [25e][38], assignment of b-peptidic
secondary structure based on CD data alone must be done with considerable
caution13). In the present case, the spectral changes observed upon dimerization of
the b-peptide might be due to aggregation, but in the absence of detailed NMR studies
other explanations cannot be ruled out.14) Aggregated peptides often show CD
spectra, that are different from those of the corresponding monomers. A good example
is the equilibrium between non-helical monomers that form ordered four-helix bundles
[39].
Experimental Part
1. General. Abbreviations: Bn: benzyl, Boc: (tert-butoxy)carbonyl, DIPEA: diisopropylethylamine
(EtN(i-Pr)2), DMAP: 4-(dimethylamino)pyridine, DMF: dimethylformamide, DTT: dithiothreitol, ESI:
electron spray ionisation, FC: flash chromatography, Fmoc: [(9 H-fluoren-9-ylmethoxy]carbonyl, Fmoc-
OSu: N-({[(9H-fluoren-9-yl)methoxy]carbonyl}oxy)succinimide, GdmCl: guanidinium hydrochloride,
HATU: O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HOBt: 1-hy-
droxy-1H-benzotriazole, HPLC: high-performance liquid chromatography, HR: high resolution, h.v.:
high vacuum (0.01 – 0.1 Torr), MALDI :matrix-assisted laser desorption ionization, MeIm: 1-methyl-1H-
imidazole, MSTN: 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, NMM: N-methylmorpholine,
13
)
)
See Sect. 7.6 in the review article [25a,b]
14
The diselenide 11c is rather poorly soluble in CD3OH, but the NMR spectrum in this solvent shows
sharp signals and well separated NH chemical shifts. This would indicate that the b-peptide 11c is
not aggregated. Aggregation normally gives rise to broadened NMR peaks (cf. [36])

Helvetica Chimica Acta – Vol. 90 (2007)
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PMBCl: para-methoxybenzyl chloride, SPS: solid-phase synthesis, TFA: trifluoroacetic acid,
(CF3COOH), TIS: triisopropylsilane ((i-Pr)3SiH), TNBS: 2,4,6-trinitrobenzene sulfonic acid. Solvents
for chromatography and workup procedures were distilled from Sikkon (anh. CaSO4; Fluka) and from
KOH (Et2O). Amino acids were purchased from Fluka or Senn. CH2N2 was prepared according to [40].
All other reagents were used as received from Fluka or Aldrich. FC: Fluka silica gel 60 (40 – 63 mm),
TLC: Merck silica gel 60 F254 plates; detection with UV, anisaldehyde soln. (9.2 ml anisaldehyde, 12.5 ml
conc. H2SO4, 3.75 ml AcOH, 340 ml EtOH), or ꢀMo-stainꢁ soln. (25 g phosphormolybdic acid, 10 g
Ce(SO4)2 · H2O, 60 ml conc. H2SO4, 940 ml H2O). Circular Dichroism (CD) Spectra: Jasco J-710
spectropolarimeter from 190 – 250 nm at 208 in 1-mm rectangular cells, all spectra were corrected for the
corresponding solvent spectrum, the molar ellipticity [q] in deg · cm2 · dmolÀ1 calculated for the
corresponding peptide. IR Spectra: Perkin-Elmer 1600 FT-IR spectrophotometer. NMR Spectra: Bruker
AMX-300 (1H: 300 MHz, 13C: 75 MHz), Varian Mercury XL 300 (1H: 300 MHz, 13C: 75 MHz); chemical
shifts d in ppm downfield from internal Me4Si (¼0 ppm); J values in Hz. MS: IonSpec Ultima 4.7 T FT
Ion Cyclotron Resonance (ICR; HR-MALDI, in 2,5-dihydroxybenzoic acid matrix) spectrometer; in
m/z (% of basis peak). Anal. and prep. RP-HPLC: Merck HPLC system (LaChrom, pump type L-7150,
UV detector L-7400, interface D7000, HPLC manager D7000) or Waters HPLC system (pump type 515,
PDA-UV detector 996 or dual wavelength detector 2487, HPLC manager Empower Pro). LC/MS was
performed on a Thermo Separation Products (TSP) HPLC system connected to a PDA detector
(UV6000LB, TSP) and to an ion-trap mass-spectrometry detector (LCQdeca, Finnigan). Linear
gradients of 5 – 60% MeCN in H2O/0.1% formic acid (0.2 ml/min) on a Waters Atlantis dC18-3 3 ꢁ 100
mm column were used. Elemental analyses were performed by the Microanalytical Laboratory of the
Laboratorium für Organische Chemie, ETH-Zürich.
2. Synthesis of the b-Amino-Acids 4 and 7. Fmoc-Sec(PMB)-OH (2) [11b,e]. NaBH4 (400 mg,
15 mmol) was added in portions to an ice-cooled soln. of selenocystine (1) (500 mg, 1.50 mmol) in 0.5n
NaOH soln. (8 ml) and EtOH (2 ml), and the mixture was stirred at r.t. until the yellow color
disappeared. After cooling again to 08, degassed 2n NaOH soln. (4 ml) and PMBCl (0.82 ml, 6.00 mmol)
were added. The mixture was stirred 30 min at 08 and 3 h at r.t. Then, the pH was adjusted to 1 – 2 with 6n
HCl, and the mixture was kept at 08 overnight. The product was collected by filtration, washed with cold
H2O, and dried under h.v. overnight. Then, a soln. of Fmoc-OSu (972 mg, 2.88 mmol) in MeCN (4 ml)
was added to an ice-cooled suspension of the crude product in a mixture of H2O (7 ml) and Et3N (0.42 ml,
2.99 mmol). After the addition of Et3N (0.42 ml, 2.99 mmol), the mixture was stirred at r.t. for 1 h. Then,
the pH was adjusted to 1 – 2 with 1n HCl, and the aq. phase was extracted with AcOEt (3 ꢁ 30 ml). The
combined org. layers were washed with 1n HCl (20 ml) and brine (20 ml), and dried (Na2SO4) and
concentrated under reduced pressure. FC (SiO2; hexane/AcOEt/AcOH 25 :25 :1) yielded 2 (1.173 g,
77%). Anal. data are in accordance with those in [11b].
Boc-Sec(PMB)-OH (5) [11b,e]. NaBH4 (400 mg, 15 mmol) was added in portions to an ice cooled
soln. of 1 (500 mg, 1.50 mmol) in 0.5n NaOH soln. (8 ml) and EtOH (2 ml), and the mixture was stirred
at r.t. until the yellow color disappeared. After cooling again to 08, degassed 2n NaOH soln. (4 ml) and
PMBCl (0.82 ml, 6.00 mmol) were added. The mixture was stirred 30 min at 08 and 4 h at r.t. Then, the
pH was adjusted to 1 – 2 and the mixture was kept at 08 overnight. The product was collected by filtration
and washed with cold H2O. After drying under h.v. overnight, the crude product was suspended in MeCN/
sat. aq. NaHCO3 soln. (20 ml/20 ml), and Boc2O (753 mg, 3.45 mmol) was added. After stirring overnight
citric acid soln. (10% in H2O, 60 ml) was added, and the aq. phase was extracted with AcOEt (3 ꢁ 50 ml).
The combined org. layers were washed with brine (2 ꢁ 50 ml), dried (MgSO4), and concentrated under
reduced pressure. FC (SiO2; hexane/AcOEt/AcOH 66 :33 :2) provided 5 (925 mg, 79%). Colorless oil. Rf
(hexane/AcOEt/AcOH 25 :25 :1) 0.48. [a]rD:t: ¼ À23.7 (c ¼ 0.5, MeOH). IR (CHCl3): 2980m, 1710s,
1610m, 1510s, 1370m, 1035m. 1H-NMR (300 MHz, CD3OD): 1.44 (s, t-Bu); 2.80 – 2.91 (m, CH2); 3.73 (s,
Me); 3.78 (s, CH2); 4.19 (t, J ¼ 6.3, CH); 6.81 (d, J ¼ 8.7, 2 arom. H); 7.20 (d, J ¼ 8.7, 2 arom. H). 13C-NMR
(75 MHz, CD3OD): 26.1, 28.0 (CH2); 29.0 (Me3C); 55.3 (CH); 55.9 (CH2); 81.0 (C); 115.1, 131.4 (CH);
132.3, 157.6, 159.9, 174.5 (C). ESI-MS: 412.2 (100, [M þ D]þ), 529.9 (23), 304.2 (38). MALDI-HR-MS:
558.0894 ([M þ Na]þ, C27H25N3O4SeNaþ; calc. 558.0903).
1-Diazo-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4-[(4-methoxybenzyl)selanyl]butan-2-
one (Fmoc-Sec(PMB)-CHN2; 3). Compound 2 (510 mg, 1.00 mmol) was dissolved in THF (5 ml) under

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Helvetica Chimica Acta – Vol. 90 (2007)
i
Ar, and the mixture was cooled to À 258. After addition of Et3N (0.15 ml, 1.07 mmol) and ClCO2 Bu
(0.14 ml, 1.07 mmol), the mixture was stirred at À 258 for 30 min. The resulting white suspension was
allowed to warm to 08, and a soln. of CH2N2 in Et2O was added until the yellow color persisted (ca.
3.00 mmol). Stirring was continued for 4 h as the mixture was allowed to warm to r.t. Excess CH2N2 was
destroyed by the addition of a few drops of AcOH and vigorous stirring for 5 min. The mixture was
diluted with Et2O (50 ml), and washed with sat. aq. NaHCO3 soln. (20 ml), sat. aq. NH4Cl (20 ml) and
brine (20 ml). The org. layer was dried (Na2SO4) and concentrated under reduced pressure. FC (SiO2;
hexane/AcOEt 2 :1 to 1:1) gave 3 (392 mg, 73%). Yellow solid. M.p. 90 – 928. Rf (hexane/AcOEt 2 :1)
0.20. [a]rD:t: ¼ À36.1 (c ¼ 0.8, CHCl3). IR (CHCl3): 3005w, 2110s. 1720s, 1640m, 1610m, 1510s, 1450m,
1
1365s, 1035m. H-NMR (300 MHz, CDCl3): 2.70 – 2.86 (m, CH2); 3.75 (br. s, CH2, Me); 4.17 (t, J ¼ 6.3,
CH); 4.32 – 4.56 (m, CH, CH2); 5.31 (s, CH); 5.72 (br. d, J ¼ 7.4, NH); 6.82 (d, J ¼ 8.5, 2 arom. H); 7.21 (d,
J ¼ 8.5, 2 arom. H); 7.30 – 7.43 (m, 4 arom. H); 7.61 (br. s, 2 arom. H); 7.77 (d, J ¼ 7.4, 2 arom. H).
13C-NMR (75 MHz, CDCl3): 24.8, 27.4 (CH2); 47.0, 54.3 (CH); 55.0 (Me); 57.1 (CH); 66.6 (CH2); 113.8,
119.8, 124.9, 126.9, 127.5, 129.8 (CH); 130.3, 141.1, 143.4, 155.5, 158.3, 193.1 (C). ESI-MS: 557.8 (100,
[M þ Na]þ), 529.9 (23), 304.2 (38). MALDI-HR-MS: 558.0894 ([M þ Na]þ, C27H25N3NaO4Seþ; calc.
558.0903).
3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-4-[(4-methoxybenzyl)selanyl]butanoic Acid
(Fmoc-b3hSec(PMB)-OH; 4). Compound 3 (392 mg, 0.73 mmol) was dissolved in THF/H2O (8 ml/
2 ml), and PhCO2Ag (34 mg, 0.15 mmol) was added with exclusion of light. The mixture was
ultrasonicated at r.t. After 4 h, H2O (20 ml) was added, and the pH was adjusted to 1 – 2 with 1n HCl. The
aq. phase was extracted with AcOEt (3 ꢁ 20 ml), and the combined org. layers were dried (Na2SO4) and
concentrated under reduced pressure. FC (SiO2; hexane/AcOEt/AcOH 66 :33 :2) yielded 4 (273 mg,
71%). White solid. M.p. 120 – 1228. Rf (hexane/AcOEt/AcOH 66 :33 :2) 0.42. [a]rD:t: ¼ À11.0 (c ¼ 0.5,
CHCl3). IR (CHCl3): 3010w, 1720s, 1610w, 1510s, 1450w, 1040m. 1H-NMR (300 MHz, CDCl3): 2.40 – 2.83
(m, 2 CH2); 3.75 (br. s, CH2, Me); 4.17 (br. s, CH); 4.22 (t, J ¼ 6.5, CH); 4.40 (br. s, CH2); 5.37 (br. d, J ¼
9.1, NH); 6.80 (d, J ¼ 7.9, 2 arom. H); 7.20 (d, J ¼ 7.9, 2 arom. H); 7.26 – 7.48 (m, 4 arom. H); 7.59 (d, J ¼ 7.3,
2 arom. H); 7.75 (d, J ¼ 7.3, 2 arom. H). 13C-NMR (75 MHz, CDCl3): 27.2, 27.7, 37.9 (CH2); 47.1, 47.8
(CH); 55.2 (Me); 66.8 (CH2); 113.9, 119.9, 125.0, 127.0, 127.7, 129.9 (CH); 130.6, 141.2, 143.7, 155.7, 158.4,
176.4 (C). MALDI-MS: 548 (100, [M þ Na]þ), 526 (16, [M þ H]þ), 346 (48). MALDI-HR-MS: 548.0938
([M þ Na]þ, C27H27NNaO5Seþ; calc. 548.0947). Anal. calc. for C27H27NO5Se (524.47): C 61.38, H 5.19, N
2.67; found: C 61.58, H 5.21, N 2.71.
1-Diazo-3-{[(tert-Butoxy)carbonyl]amino}-4-[(4-methoxybenzyl)selanyl]butan-2-one
(Boc-
Sec(PMB)-CHN2, 6). Compound 5 (1.55 g, 4.00 mmol) was dissolved in THF (20 ml) under Ar and
i
cooled to À 258. After addition of Et3N (0.60 ml, 432 mg, 4.27 mmol) and ClCO2 Bu (0.56 ml, 585 mg,
4.28 mmol), the mixture was stirred at À 258 for 30 min. The resulting white suspension was allowed to
warm to 08, and a soln. of CH2N2 in Et2O was added until the yellow color persisted (ca. 12.0 mmol).
Stirring was continued for 4 h as the mixture was allowed to warm to r.t. Excess CH2N2 was destroyed by
the addition of a few drops of AcOH and vigorous stirring for 5 min. The mixture was diluted with Et2O
(100 ml), and washed with sat. aq. NaHCO3 soln. (20 ml), sat. aq. NH4Cl (20 ml), and brine (20 ml). The
org. layer was dried (Na2SO4) and concentrated under reduced pressure. FC (SiO2; hexane/AcOEt 2 :1)
provided 6 (1.36 g, 82%). Yellow oil. Rf (hexane/AcOEt 2 :1) 0.44. [a]rD:t: ¼ À30.4 (c ¼ 0.9, CHCl3) IR
(CHCl3): 3010w, 2110s. 1710s, 1640m, 1510s, 1490s, 1370s, 1035m. 1H-NMR (300 MHz, CDCl3): 1.43 (s, t-
Bu); 2.76 (d, J ¼ 6.0, CH2); 3.76 (m, CH2, Me); 4.34 (br. s, CH); 5.33, 5.48 (2s, CH, NH); 6.81 (d, J ¼ 8.4, 2
arom. H); 7.20 (d, J ¼ 8.4, 2 arom. H). 13C-NMR (75 MHz, CDCl3): 25.1, 27.4 (CH2); 28.2 (Me3C); 54.2
(CH); 55.1 (Me); 56.8 (CH); 80.1 (C); 113.9, 129.9 (CH); 130.5, 155.0, 158.4, 192.7 (C). ESI-MS: 436.1
(100, [M þ Na]þ), 414.2 (6). MALDI-HR-MS: 436.0742 ([M þ Na]þ, C17H23N3NaO4Seþ; calc. 436.0746).
3-{[(tert-Butoxy)carbonyl]amino}-4-[(4-methoxybenzyl)selanyl]butanoic Acid (Boc-b3hSec(PMB)-
OH, 7). Compound 6 (1.24 g, 3.00 mmol) was dissolved in THF/H2O (32 ml/8 ml), and PhCO2Ag
(137 mg, 0.60 mmol) was added with exclusion of light. The mixture was ultrasonicated at r.t. After 4 h,
H2O (20 ml) and citric acid soln. (10% in H2O, 30 ml) were added, and the aq. phase was extracted with
AcOEt (1 ꢁ 100 ml, 2 ꢁ 50 ml). The combined org. layers were washed with brine (40 ml), dried
(Na2SO4), and concentrated under reduced pressure. FC (SiO2; hexane/AcOEt/AcOH 25 :25 :1) gave 7
(1.06 g, 88%). Yellow oil, which slowly crystallized to a pale yellow solid. M.p. 85 – 878. Rf (hexane/

Helvetica Chimica Acta – Vol. 90 (2007)
1663
AcOEt/AcOH 25 :25 :1) 0.54. [a]rD:t: ¼ þ8.5 (c ¼ 0.65, CHCl3). IR (CHCl3): 3010m, 1710s, 1610w, 1510s,
1370m, 1035m. 1H-NMR (300 MHz, CDCl3): 1.43 (s, t-Bu); 2.50 – 2.79 (m, 2 CH2); 3.75 (br. s, CH2, Me);
4.07 (br. s, CH); 5.14 (br. s, NH); 6.81 (d, J ¼ 8.6, 2 arom. H); 7.19 (d, J ¼ 8.6, 2 arom. H). 13C-NMR
(75 MHz, CD3OD): 21.8 (CH2); 28.4 (Me3C); 29.4, 40.1 (CH2); 49.7 (CH); 55.9 (Me); 80.4 (Me3C);
115.1, 131.4 (CH); 132.9, 157.8, 160.1, 175.0 (C). MALDI-MS: 426.1 (100, [M þ Na]þ), 404.1 (16, [M þ
H]þ). MALDI-HR-MS: 426.0783 ([M þ Na]þ, C17H25NNaO5Seþ; calc. 426.0790). Anal. calc. for
C17H25NO5Se (402.35): C 50.75, H 6.26, N 3.48; found: C 50.96, H 6.34, N 3.51.
Pentafluorophenyl 3-{[(tert-Butoxy)carbonyl]amino}-4-[(4-methoxybenzyl)selanyl]butanoate (Boc-
b3hSec(PMB)-OPFP; 7a) [35]. To a stirred ice-cooled soln. of 7 (402 mg, 1.00 mmol) and pentafluor-
ophenol (202 mg, 1.10 mmol) in dry THF (10 ml) DCC (227 mg, 1.1 mmol) was added. Stirring was
continued for 1 h at 08 and for 1 h at r.t. The suspension was cooled in an ice bath, filtered, and the filtrate
was washed with cold THF. The solvent was evaporated. The crude product was treated with Et2O
(60 ml), filtered, and the filtrate was evaporated to dryness. Recrystallization of the crude product from
hexane yielded 7a (411 mg, 72%). Light yellow solid. M.p. 1088. Rf (hexane/AcOEt 4 :1) 0.40. [a]rD:t: ¼ 3.6
(c ¼ 0.5, CHCl3). IR (CHCl3): 2980w, 1785m, 1710m, 1610w, 1520s, 1515m, 1370m, 1100m, 1005m.
1H-NMR (300 MHz, CDCl3): 1.46 (s, t-Bu); 2.69 – 2.80 (m, CH2); 2.95 – 3.06 (m, CH2); 3.78 (s, Me); 3.81
(s, Me); 4.21 (br. s, CH); 4.90 (br. s, NH); 6.82 (d, J ¼ 8.7, 2 arom. H); 7.23 (d, J ¼ 8.7, 2 arom. H). ESI-MS:
591.9 (100, [M þ Na]þ). Anal. calc. for C23H24F5NO5Se (568.40): C 48.60, H 4.26, N 2.46; found: C 48.33,
H 4.26, N 2.53.
3. Peptide Synthesis. 3.1. Fmoc Deprotection. General Procedure 1 (GP 1). The Fmoc group was
removed by treating the resin with 20% piperidine in DMF (4 ml, 3 ꢁ 10 min) under Ar bubbling. After
filtration, the resin was washed with DMF (5 ml, 4 ꢁ 1 min).
3.2. Coupling of the b-Amino Acids on Rink-Amide AM Resin. General Procedure 2 (GP 2). GP 2a:
The Fmoc deprotection was carried out according to GP 1. Solid-phase synthesis was continued by
sequential incorporation of Fmoc-protected amino acids. For each coupling step, the resin was treated
with a soln. of the Fmoc-protected b3-amino acid (3 equiv.), HATU (2.8 equiv.), and DIPEA (6 equiv.)
dissolved in the minimal amount of DMF for 1 h. Monitoring of the coupling reaction was performed
with the TNBS test [41]. In the case of a positive TNBS test (indicating incomplete coupling), the
suspension was allowed to react further for 1 h, or, after filtration, the peptide-resin was treated again
with the same Fmoc-protected amino acid (1 – 3 equiv.), and with the coupling reagents. The resin was
then filtered and washed with DMF (20 ml/g resin, 5 ꢁ 1 min) prior to the following deprotection step.
GP 2b: As described in GP 2a, but without Fmoc deprotection (GP 1).
3.3. Rink-Amide AM Resin Cleavage and Final Deprotection. General Procedure 3 (GP 3). The
cleavage from the resin and the peptide deprotection were performed according to [42]. The dry peptide-
resin was suspended in a soln. of TFA/H2O/CH2Cl2/TIS 89 :5 :5 :1 (10 ml) for 2 h. The resin was removed
by filtration, washed with TFA (2ꢁ), and the org. phase was concentrated under reduced pressure. The
resulting oily residue was treated with cold Et2O, and the formed precipitate was separated. The isolated
crude b-peptide was dried under h.v. and stored at À 208 before purification.
3.4. HPLC Analysis and Purification of b-Peptides. General Procedure 4 (GP 4). RP-HPLC Analysis
was performed on a Macherey-Nagel C18 column (Nucleosil 100-5 C18 (250 ꢁ 4 mm)) by using a linear
gradient of A: 0.1% TFA in H2O and B: MeCN, at a flow rate of 1 ml/min with UV detection at 220 nm.
tR in min. Crude products were purified by prep. RP-HPLC on a Macherey-Nagel C18 column (Nucleosil
100 – 7 C18 (250 ꢁ 21 mm) using gradient of A and B at a flow rate of 17 ml/min with UV detection at
220 nm, and then lyophilized.
H-b3hSec(PMB)-b3hAsp-b3hSer-b3hPhe-b3hLys-b3hThr-b3hVal-NH2 (8). Rink-Amide AM resin
(282 mg, 0.2 mmol, loading 0.71 mmol/g) was preswelled in DMF (10 ml) for 1 h by N2 bubbling.
Subsequently the resin was filtered. SPS was performed according to GP 2a by sequential incorporation
of Fmoc-b3hVal-OH, Fmoc-b3hThr(tBu)-OH, Fmoc-b3hLys(Boc)-OH, Fmoc-b3hPhe-OH, Fmoc-b3hSer-
OH, and Fmoc-b3hAsp(tBu)-OH. Then, the resin was Fmoc-deprotected (GP 1), washed with CH2Cl2
(4 ml, 4 ꢁ 1 min), and dried under h.v. overnight. The dried resin was divided in two equal parts (2 ꢁ
221 mg, 2 ꢁ 0.10 mmol).
The first part (221 mg, 0.10 mmol) was preswelled in DMF (10 ml) for 1 h by N2 bubbling. The resin
was filtered and Fmoc-b3hSec(PMB)-OH was coupled according to GP 2b. Then the resin was washed

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Helvetica Chimica Acta – Vol. 90 (2007)
with CH2Cl2 (4 ml, 4 ꢁ 1 min) and dried under h.v. overnight. Treatment of the peptide-resin according to
GP 3 afforded the crude peptide 8 (168 mg) as a TFA salt. Purification of this crude peptide by RP-HPLC
(30 – 95% B in 30 min: 5 min 30% B; 25 min 40% B; 30 min 95% B; tR 17.1 min) according to GP 4
yielded the TFA salt of 8 (27.1 mg) in > 98% purity.
The second part (221 mg, 0.10 mmol) was preswelled in DMF (10 ml) for 1 h by N2 bubbling. After
filtration, a mixture of Fmoc-b3hSec(PMB)-OPFP (7a) (171 mg, 0.30 mmol) and HOBt (41 mg,
0.30 mmol) in DMF was added to the resin. The suspension was mixed by N2 bubbling for 2 h.
Subsequently, the resin was filtered, washed with DMF (4 ml, 4 ꢁ 1 min), with CH2Cl2 (4 ml, 4 ꢁ 1 min),
and dried under h.v. overnight. Treatment of the peptide-resin according to GP 3 afforded the crude
peptide 8 (127 mg) as a TFA salt. Purification of this crude product by RP-HPLC (30 – 95% B in 30 min:
5 min 30% B; 25 min 40% B; 30 min 95% B; tR 17.1 min) according to GP 4 yielded the TFA salt of 8
(24.7 mg) in > 98% purity.
RP-HPLC (5 – 99% B in 60 min: 45 min 50% B, 60 min 99% B): tR 33.79, purity > 98%. MALDI-
HR-MS: 1064.5281 ([M þ H]þ, C49H78N9O12Seþ; calc. 1064.5299).
[H-b3hSec-b3hAsp-b3hSer-b3hPhe-b3hLys-b3hThr-b3hVal-NH2]2 (9). Compound 8 (25.8 mg, 20
mmol) was dissolved in 1 ml of a mixture of m-cresol, thioanisol, TFA, and TMSBr 50 :120 :750 :132
under Ar in an ice bath. After stirring for 1 h, cold Et2O was added, and the precipitate formed was
washed with cold Et2O (3ꢁ). This crude product was dried under h.v. Purification of the crude peptide by
RP-HPLC (20 – 95% B in 70 min: 5 min 20% B; 65 min 50% B; 70 min 95% B; 17 ml/min) yielded the
TFA salt of 9 (13.9 mg, 59%; tR 25.8 min) and the TFA salt of 8 (4.7 mg, 18%, tR 37.9 min).
RP-HPLC (5 – 99% B in 60 min: 45 min 50% B, 60 min 99% B): tR 28.11, purity > 98%. MALDI-
HR-MS: 1885.855 ([M þ H]þ, C82H136N18O22Se2þ ; calc. 1885.8508).
3. Selenoligation. [H-b3hAla-b3hPhe-b3hTyr-b3hGly-b3hSec-b3hAsp-b3hSer-b3hPhe-b3hLys-b3hThr-
b3hVal-NH2]2 (11c) and H-b3hAla-b3hPhe-b3hTyr-b3hGly-b3hSec(SPh)-b3hAsp-b3hSer-b3hPhe-b3hLys-
b3hThr-b3hVal-NH2 (11b). Degassed buffer (2.0 ml, 100 mm phosphate, 6m guanidinium HCl) was added
to a mixture of the peptide 10 (3.0 mg, 4.06 mmol) and the diselenide of 9 (5 mg, 2.14 mmol) in a Schlenk
flask under N2. The reaction was initated by the addition of 5% thiophenol (100 ml). The heterogeneous
mixture was vigorously stirred at r.t. Aliquots were withdrawn, diluted 10 times, and then analyzed by
HPLC and LC/MS. After the reaction was judged to be over, the mixed selenosulfide was reduced with
DTT (10 mg, 65 mmol) for 30 min, and then it was transferred to a 15-ml Falcon tube and extracted with
Et2O (2 ꢁ 10 ml). The resulting soln. was diluted with 3 ml of 50% MeCN in H2O (0.1% TFA), and that
was directly purified by prep. HPLC. The desired product was isolated as the diselenide of product 11c in
50% yield (3.4 mg). RP-HPLC (5 – 95% B in 60 min: 5 min 5% B, 50 min 60% B, 55 min 95% B): tR
35.94, purity > 99%. MALDI-HR-MS for the [A þ 12] peak: 2874.365 ([M þ H]þ; calc. for
C
136H205N26O32Seþ2 : 2874.359). Following an alternative workup where Et2O was omitted and the crude
mixture was directly injected onto the RP-HPLC, the product peak contained a large amount of
thiophenol in addition to 11b. Most of the thiophenol contaminant could be removed by lyophilization,
allowing isolation of 11b in 66% yield (5 mg). RP-HPLC as for 11c: tR 36.74, purity 90%. LC/ESI-MS:
1546.8 ([M þ H]þ, C74H108N13O16SSeþ; calc. 1546.7).
REFERENCES
[1] D. H. Holben, A. M. Smith, J. Am. Diet. Assoc. 1999, 99, 836.
[2] a) L. Li, Y. Xie, W. M. El-Sayed, J. G. Szakacs, J. C. Roberts, Life Sci. 2004, 75, 447; b) L. V. Papp, J.
Lu, A. Holmgren, K. K. Khanna, Antioxid. Redox Signaling 2007, 9, 775; c) M. P. Rayman, Lancet
2000, 356, 233.
[3] T. C. Stadtman, Annu. Rev. Biochemistry 1996, 65, 83.
[4] S. C. Gamble, A. Wiseman, P. S. Goldfarb, J. Chem. Tech. Biotechnol. 1997, 68, 123; R. F. Burk, K. E.
Hill, BioEssays 1999, 21, 231.
[5] A. Bçck, ꢀIncorporation of selenium into bacterial selenoproteinsꢁ, in ꢀSelenium in biology and
human healthꢁ, Ed. R. F. Burk, Springer-Verlag, New York, 1994, 9; A. Hüttenhofer, A. Bçck, in

Helvetica Chimica Acta – Vol. 90 (2007)
1665
ꢀRNA Structure and Functionꢁ, Cold Spring Harbor Press, 1998, 603; S. Commans, A. Bçck, FEMS
Microbiol. Rev. 1999, 23, 335.
[6] D. L. Hatfield, V. N. Gladyshev, Mol. Cell. Biol. 2002, 22, 3565.
[7] B. Nygard, Ark. Kemi 1967, 27, 341.
[8] S. Müller, H. Senn, B. Gsell, W. Vetter, C. Baron, A. Bçck, Biochemistry 1994, 33, 3404; D. Besse, F.
Siedler, T. Diercks, H. Kessler, L. Moroder, Angew. Chem., Int. Ed. 1997, 36, 883.
[9] L. Moroder, J. Pept. Sci. 2005, 11, 187.
[10] N. M. Okeley, Y. Zhu, W. A. van der Donk, Org. Lett. 2000, 2, 3603; H. Zhou, W. A. van der Donk,
Org Lett. 2002, 4, 1335.
[11] a) P. Chocat, N. Esaki, H. Tanaka, K. Soda, Anal. Biochem. 1985, 148, 485; b) T. Koide, H. Itoh, A.
Otaka, H. Yasui, M. Kuroda, N. Esaki, K. Soda, N. Fujii, Chem. Pharm. Bull. 1993, 41, 502; c) M. D.
Gieselman, L. Xie, W. A. van der Donk, Org. Lett. 2001, 3, 1331; d) A. L. Braga, P. H. Schneider,
˜
M. W. Paixao, A. M. Deobald, C. Peppe, D. P. Bottega, J. Org. Chem. 2006, 71, 4305; e) I.
Andreadou, W. M. P. B. Menge, J. N. M. Commandeur, E. A. Wortington, N. P. E. Vermeulen, J.
Med. Chem. 1996, 39, 2040; f) E. M. Stocking, J. N. Schwarz, H. Senn, M. Salzmann, L. A. Silks, J.
Chem. Soc., Perkin. Trans. 1 1997, 2443; g) D. Theodoropoulous, I. L. Schwartz, R. Walter,
Biochemistry 1967, 6, 3927; h) J. Roy, W. Gordon, I. L. Schwartz, R. Walter, J. Org. Chem. 1970, 35,
510; i) H. Tanaka, K. Soda, Methods Enzymol. 1987, 143, 240.
[12] M. Ralle, S. M. Berry, M. J. Nilges, M. D. Gieselman, W. A. van der Donk, Y. Lu, N. J. Blackburn, J.
Am. Chem. Soc. 2004, 126, 7244.
[13] L. Moroder, H. J. Musiol, N. Schaschke, L. Chen, B. Hargittai, G. Barany, in ꢀ Houben-Weyl,
Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimeticsꢁ (E22a), M. Goodman,
A. Felix, L. Moroder, C. Toniolo, Eds., Georg Thieme Verlag, Stuttgart, 2002, 384 – 423.
[14] L. Moroder, F. Marchiori, G. Borin, E. Scoffone, Biopolymers 1973, 12, 493.
[15] K. M. Harris, S. Flemer Jr., R. J. Hondal, J. Pept. Sci. 2007, 13, 81.
[16] D. Besse, L. Moroder, J. Pept. Sci. 1997, 3, 442.
[17] L. Moroder, D. Besse, H.-J. Musiol, S. Rudolph-Bçhner, F. Siedler, Biopolymers (Pept. Sci.) 1996, 40,
207.
[18] T. Wieland, E. Bokelmann, L. Bauer, H. U. Lang, H. Lau, Liebigs Ann. Chem. 1953, 583, 129; D. S.
Kemp, S. L. Leung, D. J. Kerkman, Tetrahedron Lett. 1981, 22, 181; P. E. Dawson, T. W. Muir, I.
Clarke-Lewis, S. B. H. Kent, Science 1994, 266, 776; T. Kimmerlin, D. Seebach, J. Pept. Res. 2005, 65,
229.
[19] M. D. Gieselman, Y. Zhu, H. Zhou, D. Galonic, W. A. Van der Donk, ChemBioChem 2002, 3,
709.
[20] R. J. Hondal, B. L. Nilsson, R. T. Raines, J. Am. Chem. Soc. 2001, 123, 5140.
[21] R. Quaderer, A. Sewing, D. Hilvert, Helv. Chim. Acta 2001, 84, 1197.
[22] R. Quaderer, D. Hilvert, Chem. Commun. 2002, 2620.
[23] a) T. W. Muir, D. Sondhi, P. A. Cole, Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705; b) T. C. Evans Jr., J.
Brunner, M.-Q. Xu, Protein Sci. 1998, 7, 2256; T. W. Muir, Annu. Rev. Biochem. 2003, 72, 249.
[24] S. M. Berry, M. D. Gieselman, M. J. Nilges, W. A. van der Donk, Y. Lu, J. Am. Chem. Soc. 2002, 124,
2084.
[25] a) D. Seebach, A. K. Beck, D. J. Bierbaum, Chem. Biodiv. 2004, 1, 1111; b) D. Seebach, A. K. Beck,
D. J. Bierbaum, ꢀDie Welt der b- und g-Peptide aus homologisierten proteinogenen Aminosäuren
und anderen Bausteinenꢁ, Verlag Helvetica Chimica Acta AG, Zürich, 2004; c) R. P. Cheng, S. H.
Gellman, W. F. DeGrado, Chem. Rev. 2001, 101, 3219; d) D. Seebach, M. Overhand, F. N. M. Kühnle,
B. Martinoni, L. Oberer, U. Hommel, H. Widmer, Helv. Chim. Acta 1996, 79, 913; e) D. Seebach,
J. V. Schreiber, P. I. Arvidsson, J. Frackenpohl, Helv. Chim. Acta 2001, 84, 271; f) D. Seebach, S.
Abele, K. Gademan, B. Jaun, Angew. Chem. 1999, 111, 1700; Angew. Chem., Int. Ed. 1999, 38, 1595.
[26] H. Wiegand, B. Wirz, A. Schweitzer, G. Gross, M. I. Rodriguez Perez, H. Andres, T. Kimmerlin, M.
Rueping, D. Seebach, Chem. Biodiv. 2004, 1, 1812; H. Wiegand, B. Wirz, A. Schweitzer, G. P.
Camenisch, M. I. Rodriguez Perez, G. Gross, R. Woessner, R. Voges, P. I. Arvidsson, J. Frackenpohl,
D. Seebach, Biopharm. Drug Dispos. 2002, 23, 251.
[27] F. Arndt, B. Eistert, Ber. Dtsch. Chem. Ges. 1935, 68, 200.

1666
Helvetica Chimica Acta – Vol. 90 (2007)
[28] a) J. Podlech, D. Seebach, Angew. Chem. 1995, 107, 622; Angew. Chem., Int. Ed. 1995, 34, 471; b) J.
Podlech, D. Seebach, Liebigs Ann. Chem. 1995, 1217; c) G. Lelais, P. Micuch, D. Josien-Lefebvre, F.
Rossi, D. Seebach, Helv. Chim. Acta 2004, 87, 3131.
[29] A. Müller, C. Vogt, N. Sewald, Synthesis 1998, 837.
[30] L. Wolff, Liebigs Ann. Chem. 1912, 394, 23.
[31] H. Takada, P. Metzner, C. Philouze, Chem. Commun. 2001, 2350.
[32] V. K. Aggarwal, J. Richardson, Chem. Commun. 2003, 2644.
[33] W. Kirmse, Eur. J. Org. Chem. 2002, 2193.
[34] T. Ye, A. McKervey, Chem. Rev. 1994, 94, 1091.
[35] L. Kisfaludy, I. Schçn, Synthesis 1984, 325.
[36] T. Kimmerlin, D. Seebach, D. Hilvert, Helv. Chim. Acta 2002, 85, 1812.
´
[37] H. G. Viehe, R. Merenyi, L. Stella, Z. Janousek, Angew. Chem., Int. Ed. 1979, 18, 917.
ˇ
[38] T. Kimmerlin, D. Seebach, Helv. Chim. Acta 2003, 86, 2098; D. Seebach, T. Kimmerlin, R. Sebesta,
M. A. Campo, A. Beck, Tetrahedron 2004, 60, 7455; E. J. Petersson, C. J. Craig, D. S. Daniels, J. X.
Qiu, A. Schepartz, J. Am. Chem. Soc. 2007 129, 5344 and ref cit. herein.
[39] R. Fairman, H.-G. Chao, T. B. Lavoie, J. J. Villafranca, G. R. Matsueda, J. Novotny, Biochemistry
1996, 35, 2824; C. Vu, J. Robblee, K. M. Werner, R. Fairman, Protein Sci. 2001, 10, 631.
[40] J. Podlech, D. Seebach, Liebigs Ann. Chem. 1995, 1217.
[41] W. S. Hancock, J. E. Battersby, Anal. Biochem. 1976, 71, 260.
[42] Novabiochem 2002/3 Catalogue: The Fine Art of Solid Phase Synthesis.
Received July 18, 2007