Lipidation of Cysteine or Cysteine-Containing Peptides Using the Thiol-Ene Reaction (CLipPA)

Article abstract of DOI:10.1002/ejoc.201501375

The efficient synthesis of N^α-protected S-palmitoylated cysteine building blocks using thiol-ene coupling is described. These building blocks were incorporated into a resin-bound peptide under racemisation-suppressing conditions, and the degree of racemisation during the coupling process was assessed. The direct conjugation of vinyl palmitate with the sulfhydryl side-chain of a cysteine residue on a semiprotected peptide was also studied. The reaction gave both mono- and bispalmitoylated cysteine residues in varying proportions, depending on the reaction conditions adopted. The S-selective lipidation of cysteine or cysteine-containing peptides has been optimised to produce an S-palmitoylated building block or an S-palmitoylated peptide directly with high conversions and excellent yields. Key to this process, for which we coin the term "CLipPA", is the use of tBuSH together with triisopropylsilane as radical quenchers to favour the formation of monolipidated products.

Full text of DOI:10.1002/ejoc.201501375

DOI: 10.1002/ejoc.201501375
Full Paper
Peptide Lipidation
Lipidation of Cysteine or Cysteine-Containing Peptides Using
the Thiol-Ene Reaction (CLipPA)
Sung-Hyun Yang,[a,b] Paul W. R. Harris,[a,b,c] Geoffrey M. Williams,[a,b,c] and
Margaret A. Brimble*[a,b,c]
Abstract: The efficient synthesis of Nα-protected S-palmitoyl- assessed. The direct conjugation of vinyl palmitate with the sulf-
ated cysteine building blocks using thiol-ene coupling is de- hydryl side-chain of a cysteine residue on a semiprotected
scribed. These building blocks were incorporated into a resin- peptide was also studied. The reaction gave both mono- and
bound peptide under racemisation-suppressing conditions, and bispalmitoylated cysteine residues in varying proportions, de-
the degree of racemisation during the coupling process was pending on the reaction conditions adopted.
Introduction
purity. These compounds can be used directly in solid-phase
peptide synthesis to construct lipopeptides. We also report im-
proved conditions for the highly selective mono S-palmitoyla-
tion of immunogenic peptide sequences to give lipopeptides
in high yield and purity.
The hydrothiolation of an unsaturated system, known as thiol-
ene coupling, is a thermally or photochemically induced trans-
formation that occurs by radical-promoted alkylation of a thiol
with an unsaturated moiety, usually an alkene.[1] It has found
particular utility in the fields of synthetic polymers,[2] synthetic
chemistry, and bioconjugation,[3] owing to its atom economy,
wide tolerance of functional groups, and straightforward reac-
tion conditions. We have previously reported a successful appli-
cation of the thiol-ene reaction to the efficient synthesis of self-
adjuvating peptides by monopalmitoylation of fluorenylmethy-
loxycarbonyl (Fmoc)-cysteinyl or cysteinyl peptides with vinyl
palmitate.[4] These monoacyl lipopeptide constructs, obtained
in a single chemical step, have been shown to have immuno-
genic activity comparable to that of the corresponding, more
synthetically demanding, less tractable, and more expensive
Pam3Cys motif. However, although the monoacyl lipopeptides
were prepared in adequate quantities for biological evaluation,
the overall yields and/or conversions in this initial study were
unsatisfactory, thereby limiting their use as building blocks for
more complex systems.
Results and Discussion
Lipidation of Nα-Protected Cysteines
We originally reported that vinyl palmitate (1) underwent the
thiol-ene reaction with Nα-protected Fmoc-cysteine 2 after irra-
diation at 365 nm for 60 min with the initiator 2,2-dimethoxy-
2-phenylacetophenone (DMPA) to give thioether 5 in moderate
yield (44 %)[4a] (Scheme 1). Monitoring the reaction by HPLC
however, revealed that 2 was completely consumed, which sug-
gests that the reaction parameters and purification method
could be improved to increase the yield of 5.
We now coin the term “CLipPA” — Cysteine Lipidation on a
Peptide or Amino acid — to describe this reaction, and in this
paper we report a detailed study of the CLipPA reaction be-
tween vinyl palmitate and Nα-protected cysteine derivatives,
which gives the S-palmitoylated compounds in high yield and
[a] School of Chemical Sciences, The University of Auckland,
23 Symonds St., Auckland 1142, New Zealand
Scheme 1. Synthesis of Nα-protected S-palmitoylated cysteines 5–7.
E-mail: m.brimble@auckland.ac.nz
http://www.auckland.ac.nz/
[b] School of Biological Sciences, The University of Auckland,
3A Symonds St, Auckland 1142, New Zealand
[c] Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological
Sciences, The University of Auckland,
In this work, we sought to modify the Nα-protecting group,
the radical initiator, and the activation method in order to de-
fine optimal conditions to generate the S-palmitoylated Nα-pro-
tected cysteines in good yield. This synthetic goal is important
as we envisaged that building blocks 5, 6, and 7, prepared from
appropriately protected cysteines 2, 3, or 4, could be used di-
rectly in routine Fmoc or Boc (tert-butoxycarbonyl) solid-phase
Auckland 1142, New Zealand
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/ejoc.201501375.
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peptide synthesis through acylation of any resin-bound amine. 4 was quantitatively converted to S-palmitoylated product 7
In order to ensure efficient coupling to the solid-phase resin, under microwave irradiation (Table 1, entry 13) using AIBN as
sufficient quantities of the amino acid building block are ini- initiator, although other methods such as using UV light
tially required, hence the need for a robust synthetic method (Table 1, entries 10 and 11) or thermal heating in 1,2-dichloro-
to access these S-palmitoylated building blocks.
ethane (Table 1, entry 12) also gave excellent yields (87–95 %)
of 7. In all cases examined (Table 1), it was necessary to use the
Nα-protected cysteine; attempting the thiol-ene reaction di-
rectly on native, unprotected cysteine using UV light and DMPA
for 1 h gave none of the expected S-palmitoylated product.
Nα-Protected Fmoc, tert-butyloxycarbonyl (Boc) or N-acetyl-
ated (Ac) cysteines 2, 3, and 4 were treated with an excess
of vinyl palmitate in the presence of DMPA[5] or 2,2-azo-bis(2-
methylpropionitrile)[6] (AIBN) in either dichloromethane or 1,2-
dichloroethane. The reactions were carried out under thermal
heating, microwave irradiation, or UV light, and the product(s)
were purified directly by silica gel chromatography. Purification
by reverse-phase HPLC resulted in poor recoveries, probably
due to the hydrophobic nature of 5, 6, and 7. The results are
summarised in Table 1.
Coupling of Nα-Protected S-Palmitoylated Cysteines 5 and
7 to Resin-Bound Peptide 8
With S-palmitoylated cysteine building blocks in hand, we went
on to examine the solid-phase coupling of Fmoc-protected 5 or
Ac-protected 7 to a model resin-bound peptide 8. The peptide
sequence SKKKK-NLVPMVATV-OH was chosen as a model pept-
ide for this study. The sequence is derived from a truncated
peptide epitope from the cytomegalovirus (CMV) ppUL83 pro-
tein, which is known to stimulate CD8+ cytotoxic T-cells.[8] This
peptide has previously been used in our group for the incorpo-
ration of a palmitoyl functionality using either copper(I)-cata-
lysed azide–alkyne cycloaddition “CuAAC” chemistry[9] or a
thiol-ene[4a] reaction. The SKKKK sequence was included as the
four lysine residues greatly improve the solubility and tractabil-
ity of the lipopeptides, and, when conjugated with palmitoyl-
ated cysteine, the motif has established itself as a potent TLR2
agonist.[10] Resin-bound peptide 8 was synthesised with the
Met residue substituted for a Cys(tBu) in order to demonstrate
that a suitably protected sulfhydryl side-chain of a cysteine resi-
due could be tolerated by the thiol-ene reaction conditions.
Side-chain unmasking then gives a handle for further manipula-
tion at this cysteine residue.
Table 1. Optimisation of the thiol-ene reaction using Fmoc-Cys 2, Boc-Cys 3,
or Ac-Cys 4 with vinyl palmitate (1).
Entry
Initiator
Conditions[a]
Yield[c]
1
2
3
4
2
2
2
2
AIBN (1.0 equiv.)
AIBN (0.5 equiv.)
DMPA (0.2 equiv.) hν (365 nm), 60 min
μwave, 100 W, 70 °C, 80 min 5 (41 %)
reflux, 90 °C, 150 min[b]
5 (55 %)
5 (67 %)
5 (82 %)
DMPA (1 equiv.)
hν (365 nm), 60 min
5
6
7
8
3
3
3
3
AIBN (0.5 equiv.)
AIBN (1 equiv.)
DMPA (0.2 equiv.) hν (365 nm), 60 min
DMPA (1 equiv.) hν (365 nm), 60 min
reflux, 90 °C, 40 min[b]
μwave, 100 W, 70 °C, 80 min 6 (77 %)
6 (54 %)[b]
6 (78 %)
6 (82 %)
9
4
4
4
4
4
DMPA (0.2 equiv.) hν (365 nm), DTT, 60 min
DMPA (0.2 equiv.) hν (365 nm), 60 min
7 (74 %)
7 (87 %)
7 (88 %)
7 (95 %)
10
11
12
13
DMPA (1. eq.)
AIBN (0.5 equiv.)
AIBN (1 equiv.)
hν (365 nm), 60 min
reflux, 90 °C, 40 min[b]
μwave, 100 W, 70 °C, 80 min 7 (>99 %)
[a] CH2Cl2 as solvent. [b] Cl-(CH2)2-Cl as solvent. [c] Isolated yield after flash
column chromatography on silica gel.
Fmoc-Cys-OH 2 underwent smooth conversion to S-palmi-
toylated product 5 (Table 1, entries 1–4), optimally using UV
light and an excess of the radical initiator DMPA giving the
desired product 5 in 82 % isolated yield (Table 1, entry 4). The
use of conventional heating or microwave irradiation gave
lower yields of 41 or 55 %, respectively (Table 1, entries 1 and
2), perhaps due to premature cleavage of the Nα-Fmoc protect-
ing group. Boc-Cys-OH 3 was converted to S-palmitoylated
product 6 under UV irradiation with an excess of DMPA in an
isolated yield of 82 % after 1 h (Table 1, entry 8). Carrying out
the reaction under thermal heating or microwave irradiation
with AIBN as the radical initiator resulted in lower yields
(Table 1, entries 5 and 6). This may be due to the instability of
the tert-butyloxycarbonyl group, which may undergo de-tert-
butylation and subsequent loss of CO2 at high temperatures.
Peptidyl resin 8 was synthesised by elongation of the pept-
ide chain using Fmoc-SPPS (solid-phase peptide synthesis), with
the reagents HATU {1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate}, diiso-
propylethylamine (DIPEA), and piperidine in DMF (20 % v/v), at
room temperature. Activation and coupling of cysteine residues
without epimerisation is recognised to be problematic in SPPS,
and is influenced by the choice of coupling reagent, base, sol-
vent conditions, and the protecting group on the sulfhydryl
side-chain.[11] Coupling of S-palmitoylated cysteine building
block 5 or 7 was therefore achieved using PyBOP [(benzotri-
azol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate;
5 equiv.] and 2,4,6-trimethylpyridine (TMP; 5 equiv.) at room
temperature, conditions known[12] to suppress racemisation
(Scheme 2). The choice of Nα-protecting group for the amino
We next examined the thiol-ene chemistry using Ac-Cys-OH acid may also affect the degree of racemisation that occurs dur-
4, which bears an amide on the α-nitrogen (Table 1, entries 9– ing the formation of the activated ester.[12a] To examine this
13). It has been shown that an N-acetyl group on the cysteine effect, resin-bound peptide 8 was either derivatised with 5, and
α-nitrogen plays an important role in modulating the immuno- the Fmoc protecting group exchanged for an acetyl group be-
genic potency of an S-palmitoylated cysteinyl-serine (Cys-Ser) fore cleavage to give peptide 9, or building block 7 was cou-
derivative.[7] We also reasoned that the acetamide moiety may pled to give 9 directly. Comparison of the HPLC traces revealed
be more stable to the reaction conditions than the Fmoc and that a 1:1 ratio of epimers was formed when acetamide-pro-
Boc carbamate-based protecting groups. In contrast to the re- tected building block 7 was used in the coupling reaction
sults obtained from Nα-protected cysteines 2 and 3, Ac-Cys-OH (Scheme 2, chromatogram c), whereas no detectable racemisa-
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Scheme 2. Coupling of Nα-protected S-palmitoylated cysteine building blocks 5 or 7 with resin-bound peptide 8. a) Iterative Fmoc-SPPS: i) deprotection of
Nα-Fmoc group: piperidine/DMF (20 % v/v), room temp., 5 min × 2; ii) Coupling of Fmoc-AA-OH: Fmoc-AA-OH (5 equiv.), HATU (4.6 equiv.), DIPEA (6 equiv.),
room temp., 40 min; b) i) 5 or 7, PyBOP (5 equiv.), 2,4,6-trimethylpyridine (5 equiv.) room temp.; For building block 5: ii) piperidine/DMF (20 % v/v) then Ac2O/
DMF (20 % v/v); iii) TFA/DODT/H2O/TIPS (94:2.5:2.5:1 v/v, resin cleavage); c) HPLC chromatogram of crude 9 synthesised using building block 7; d) HPLC
chromatogram of crude 9 synthesised using building block 5. Inset: observed ESI-MS spectrum of the main peak (calcd. mass: 1998.2; found 1999.3 [M + H]+,
1000.1 [M + 2H]2+, 667.0 [M + 3H]3+, 500.5 [M + 4H]4+). Chromatographic separations were carried out using a linear gradient of 5–95 % B over 30 min, ca.
3 % B per min. Buffer A: H2O containing TFA (0.1 % v/v); Buffer B: acetonitrile containing TFA (0.1 % v/v).
tion was observed when Fmoc-protected building block 5 was terminus was acetylated. The peptide was liberated from the
used (Scheme 2, chromatogram d). Thus, even though the cou- solid support by cleavage with cocktail A[13] (TFA/DODT/H2O/
plings of 5 and 7 both proceeded to completion, Fmoc-pro- TIPS, 94:2.5:2.5:1 v/v), and then purified by HPLC, ready for lipi-
tected building block 5 is clearly a more versatile reagent.
dation.
Direct Lipidation on a Semiprotected Peptide 10 Bearing a
Free Cysteine Sulfhydryl Side-Chain
Optimisation of Reaction Solvent
Firstly, we examined the viability of the reaction between vinyl
We next examined a more convergent approach for the synthe- palmitate (1) and peptide 10 using the polar organic solvents
sis of the desired product (i.e., 9). This involved using the thiol- N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and
ene reaction to install the palmitoyl group onto the side-chain N-methyl-2-pyrrolidone (NMP) in an effort to fully solvate both
of a cysteine residue by direct conjugation of vinyl palmitate the hydrophilic peptide and hydrophobic vinyl palmitate (1).
(1) with fully formed peptide 10 (Scheme 3). This strategy has The use of DMSO as solvent was discounted owing to the low
been briefly reported by our group,[4] and we sought to further solubility of vinyl palmitate (1), which led to low conversion of
extend our understanding of this transformation. As a starting peptide 10 into the desired mono adduct (i.e., 9) (data not
point, peptide 10, bearing a free sulfhydryl side-chain, was syn- shown). DMF proved a more suitable solvent in that all the
thesised using Fmoc-SPPS as described previously, and the N- reagents were soluble, and efficient conversion to product 9
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Scheme 3. CLipPA direct conjugation of vinyl palmitate (1) and semiprotected peptide 10. a) Iterative Fmoc-SPPS: i) Deprotection of Nα-Fmoc group: piperidine/
DMF (20 % v/v), room temp., 5 min × 2; ii) Coupling of Fmoc-AA-OH: Fmoc-AA-OH (5 equiv.), HCTU (4.6 equiv.), DIPEA (6 equiv.), room temp., 40 min;
iii) piperidine/DMF (20 % v/v), room temp., 5 min × 2; then Ac2O/DMF (20 % v/v) room temp., 10 min; iv) Cleavage of peptidyl resin: Cocktail A (TFA/DODT/
H2O/TIPS, 94:2.5:2.5:1 v/v), 2 h, room temp.; b) DMPA (0.5 equiv.), NMP, TFA (5 % v/v), additives (see Table 2), UV (365 nm), room temp. DODT = 2,2′-
(ethylenedioxy)diethane dithiol; HCTU = 1-[bis(dimethylamino)methylene]-5-chlorobenzotriazolium 3-oxide hexafluorophosphate; TIPS = triisopropylsilane;
TFA = trifluoroacetic acid.
was observed. However, the heat generated from mixing DMF Addition of TFA and tert-Butylthiol
and residual TFA (vide infra) induced the formation of a sulfox-
The first lipidation of semiprotected peptide 10 and vinyl palmi-
tate (1) was carried out using DMPA as initiator, but excluding
other additives (Table 2, entry 1). After irradiating the mixture
for 30 min with UV light, a sample was analysed by LC–MS.
This showed, in addition to DMPA degradation products (see
Supporting Information Figure S2), a moderate conversion
(58 %) of peptide 10 to the desired mono adduct (i.e., 9; 84 %
of product) and bis adduct 12 (16 % of product) (Figure 1).
The formation of 12 arises from reaction of carbon-centred ꢀ-
sulfanylalkyl radical intermediate 11 with a second equivalent
of vinyl ester (Scheme 3); this process — telomerisation — oc-
curs in competition with the expected quenching of 11 by a
thiol leading to mono adduct 9 and propagation of the thiol-
ene reaction.
ide (+16 Da) by-product, thus complicating HPLC analysis and
product recovery. We assume that this results from oxidation of
the thioether moiety of the palmitoylated cysteine residue
present in 9. NMP was finally determined to be most suitable
solvent for this transformation, and was therefore used exclu-
sively for the subsequent optimisation studies.
Optimisation of Radical Quencher
Initially, we used our original reaction conditions[4a] to effect
direct conjugation of vinyl palmitate (1) with semiprotected
peptide 10. The procedure entailed irradiating at 365 nm a solu-
tion of the radical initiator DMPA (0.5 equiv.) and the peptide
in NMP as the solvent, with dithiothreitol (DTT; 3 equiv.) in-
cluded as an exogenous thiol source. We had previously con-
cluded that extraneous thiols were required to improve the
product profile; they presumably decrease unwanted side-reac-
tions such as the formation of telomers or mixed disulfides by
facilitating the hydrogen abstraction process to quench the in-
termediate radical species (e.g., 11) and give the desired prod-
uct.[14] We have previously used various thiols such as reduced
glutathione (GSH), 2,2′-(ethylenedioxy)diethanethiol (DODT),
and (optimally) DTT. However, in this work, careful examination
of the LC–MS profile of the reaction between peptide 10 and
vinyl palmitate (1) revealed the formation of by-products with
masses of 120 and 152 Da greater than product 9, indicative of
the addition of DTT and fragments thereof to carbon-centred
radical 11.[15]
An improved RP-HPLC profile was observed when trifluoro-
acetic acid (TFA; 5 % v/v) was added[14,17] (see Supporting Infor-
mation Figure S4) to the reaction mixture; this had a minimal
effect on the conversion rate. Decreasing the pH of the reaction
mixture may suppress the formation of by-products by ensuring
protonation of the electron rich side-chains of residues such as
lysine, arginine, and histidine, which could otherwise partici-
pate in single-electron transfers and form radical species during
the propagation step.
Next, the effect of adding of both tert-butylthiol and TFA
to the reaction mixture was investigated. While addition of an
exogenous thiol slightly favoured an increase of mono adduct
9 over bis adduct 12, low conversion (<70 %) of peptide 10
We therefore substituted DTT with the hindered tert-butyl- was still problematic (Table 2, entry 2). Extending the reaction
thiol (tBuSH); this reagent should still act as an efficient chain- time to 60 min under identical reaction conditions did not alter
transfer agent, but its steric bulk should prevent the formation the conversion of peptide 10 into products 9 and 12 (see Sup-
of S-alkylation by-products.[16] Gratifyingly, this proved to be porting Information Figure S3); in fact, the reaction appeared
the case, as no analogous by-products were detected by LC– to stop within the initial 30 min of UV irradiation, and could
MS. Thus, tert-butylthiol was preferentially used in subsequent not be driven to completion, even upon the addition of a sec-
experiments.
ond portion of both DMPA and vinyl palmitate (1). These pre-
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Table 2. Optimisation of lipidation. Conjugation of peptide 10 and vinyl palmitate (1) in NMP[a] using DMPA[b] as radical initiator.
Entry
Vinyl palmitate (1) [equiv.][c]
tBuSH [equiv.][c]
TIPS [equiv.][c]
Conversion[f] [%]
Products[f]
1
2
3
4
5
6
7
8
7
7
0
3
3
0
0
0
0
58
69
84
93
94
88
94
78
60
81
92
90
26
91
9 (84 %) 12 (16 %)
9 (97 %) 12 (3 %)
9 (65 %) 12 (35 %)
9 (76 %) 12 (24 %)
9 (88 %) 12 (12 %)
9 (95 %) 12 (5 %)
9 (95 %)[g] 12 (5 %)
9 (67 %) 12 (33 %)
9 (98 %) 12 (2 %)
9 (> 99 %) 12 (<1 %)
9 (97 %) 12 (3 %)
9 (95 %) 12 (5 %)
9 (> 99 %) 12 (trace)
9 (96 %) 12 (4 %)
70
70
70
70
70
70
7
20
35
100
70
70
80
80
40
80
0
80
80
80
80
80
80
40
40
80
80
80
80
80
80
80
80
9
10
11
12
13[d]
14[e]
[a] 30 min reaction time, with 5 % TFA based on final reaction volume. [b] 0.5 equiv. relative to peptide 10. [c] Molar equivalent relative to peptide 10.
[d] Dimethyl sulfoxide as solvent. [e] N,N′-Dimethylformamide as solvent. [f] Conversion of peptide 10, mono adduct 9, and bis adduct 12 is based on
integration of the corresponding peaks on the RP-HPLC profile at 210 nm. The relative amounts of 9 and 12 are cited as percentages. [g] 72 % isolated yield
after RP-HPLC purification.
erated, and this may contribute to the low conversion observed
in this case. We postulated that a higher conversion could be
achieved by adding a larger excess of vinyl palmitate (1), as this
would facilitate an increase in the rate of formation of carbon-
centred radical intermediate 11, and thence form mono adduct
9 via chain transfer with the hydrogen radical source. Indeed,
upon addition of a large excess of vinyl palmitate (1; 70 equiv.),
the overall conversion of peptide 10 increased markedly to
84 % (Table 2, entry 3, see Supporting Information Figure S6).
However, this was accompanied by a corresponding increase in
the proportion of bis adduct 12 to 35 %. It is interesting to
note that despite the high concentration of vinyl palmitate, no
higher-order propagations were observed.[14] Increasing the
amount of tert-butylthiol (Table 2, entry 4, see Supporting Infor-
mation Figure S7) enhanced the conversion of 10 further to
93 %, and diminished the proportion of 12 to 24 %. Presumably,
this resulted from shifting the balance of competing pathways
available to radical intermediate 11 — namely, reversibility, tel-
omerisation and quenching — in favour of the radical-quench-
ing route that leads to 9.
Addition of a Coreductant
Figure 1. Lipidation reaction between vinyl palmitate (1) and peptide 10 us-
ing DMPA and UV light. a) HPLC chromatogram of Table 2, entry 1, t = 0 min;
We next envisaged that the formation of bis adduct 12 could be
Inset: observed ESI-MS spectrum of the main peak for peptide 10 (calcd.
suppressed by the introduction of a coreductant to the reaction
mixture that facilitates more rapid hydrogen-radical abstraction
from the coreductant to the carbon-centred radical intermedi-
ate 11, thus giving the desired monopalmitoylated adduct 9.
Organosilanes are radical-based reducing agents whose
hydrogen-donor ability can be fine-tuned by varying the sub-
stituents on the silicon atom.[19] Thus, judicious selection of the
organosiliane reagent may induce the formation of the desired
product (i.e., 9) by fast hydrogen transfer to the carbon-centred
radical intermediate 11.
mass: 1715.0; found 858.4 [M + 2H]2+, 572.5 [M + 3H]3+, 429.6 [M + 4H]4+);
b) HPLC chromatogram of Table 2, entry 1, t = 30 min; c) HPLC chromatogram
of Table 2, entry 8, t = 30 min; d) HPLC chromatogram of Table 2, entry 7,
t = 30 min; Inset: observed ESI-MS spectrum of the main peak for mono
adduct 9 (calcd. mass: 1998.2; found 999.9 [M + 2H]2+, 667.0 [M + 3H]3+
,
500.6 [M + 4H]4+); for bis adduct 12 (calcd. mass: 2280.6; found 1141.1 [M +
2H]2+, 761.1 [M + 3H]3+, 571.1 [M + 4H]4+); Chromatographic separations were
carried out using a linear gradient of 5–95 % B over 30 min, ca. 3 % B per
min. Buffer A: H2O containing TFA (0.1 % v/v); Buffer B: acetonitrile containing
TFA (0.1 % v/v).
liminary observations suggested that a rapid conversion of
We chose readily available triisopropylsilane (TIPS) as the cor-
peptide 10 into product 9 is required in order to maximise eductant (40 equiv.) for the thiol-ene reaction (Table 2, entry 5,
conversion of the starting peptide. It is well known that the first see Supporting Information Figure S7). Gratifyingly, using TIPS
step, in which carbon-centred radical intermediates such as 11 as a coreductant, the formation of bis adduct 12 was substan-
are formed, is reversible;[18] thus, the olefin and thiol are regen- tially diminished to 12 %, while peptide 10 was consumed with
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Table 3. Conjugation of peptides 13 and 14 with vinyl palmitate (1) to form monopalmitoylated product under unoptimised[a] and optimised[b] conditions.
Entry
Peptide sequence
Unoptimised
Optimised
conditions [%][e]
conditions [%][e]
1
2
Peptide 13[c]
4
81
46
Ac-CSKKKKGARGPESRLLE-FYLAMPFATPMEAELARRSLAQDAPPL-OH
Peptide 14[d]
<1
H2N-CSKKKKVPGVLLKEFTV-SGNILTIRLTAADHR-OH
[a] DMPA (0.5 equiv.), vinyl palmitate (7 equiv.) in NMP. [b] DMPA (0.5 equiv.), vinyl palmitate (70 equiv.), tert-butylthiol (80 equiv.), triisopropylsilane (80 equiv.),
TFA (5 % v/v) in NMP. [c] Derived from NY-ESO-1 [79-116]. [d] Derived from NY-ESO-1 [118-143]. [e] Conversion of peptides 13 and 14 to corresponding
monopalmitoylated product S1 and S2 is based on the integration of corresponding peaks on RP-HPLC profile at 230 nm.
>95 % conversion, and the desired product (i.e., 9) was formed product mixture was reduced with tetrabutylammonium iodide
in 88 % yield. A slight improvement in the conversion of pept- in trifluoroacetic acid to simplify HPLC analysis.[20]
ide 10 or the ratio of product 9 to 12 was observed when the
amounts of tert-butylthiol and TIPS were varied slightly (Table 2,
entries 6 and 7).
Conclusions
When TIPS was used as a sole reductant (without tBuSH) in
the reaction, a large amount of bispalmitoylated by-product 12
was observed (Table 2, entry 8, see Supporting Information Fig-
ure S7). This experimental observation indicated that while tert-
butylthiol or TIPS can be used as a sole hydrogen donor source
for the radical chain process, a synergistic effect between these
two reductants minimises the formation of 12.
In conclusion, Nα-protected-cysteinyl building blocks 5–7, func-
tionalised with an S-palmitoyl fatty chain, were synthesised in
high yield using a thiol-ene radical conjugation reaction. These
pre-formed building blocks were directly incorporated into a
peptide sequence using Fmoc-SPPS. Significant epimerisation
was observed when coupling AcNH-Cys(palmitoyl)-OH 7, but
this did not take place with carbamate FmocNH-Cys(palmitoyl)-
OH (5). Direct conjugation of the palmitoyl group onto the
cysteine residue of unprotected peptide 10 using a thiol-ene
reaction was also demonstrated. The key reagents for effective
conjugation of peptide 10 with vinyl palmitate (1) were tert-
butylthiol and triisopropylsilane, acting as dual hydrogen-donor
components. The use of this dual reductant system effected
high conversion of starting peptide 10, giving mono adduct 9
as the major component, and minimising formation of bis ad-
duct 12. Our optimised conditions were applied to longer pept-
ides 13 and 14, which exclusively gave monopalmitoylated ad-
ducts in excellent to good yields that could not be accessed
without the addition of tert-butylthiol and triisopropylsilane. We
believe that the experimental conditions that have been care-
fully optimised in this study will be generally applicable as an
efficient method for the site-specific lipidation of cysteine-con-
taining polypeptides (CLipPA technology).
Final Comments
Lastly, having established the efficacy of the thiol and silane as
a reducing pair, the relative quantity of vinyl palmitate (1) was
progressively decreased to assess the effect of this on the effi-
ciency of the reaction (Table 2, entries 9–12, see Supporting
Information Figure S8). Little difference in conversion was ob-
served for a 100- or 35-fold excess of 1; however, a progressive
decrease in the conversion of peptide 10, from 81 to 60 %, was
noted as the levels of 1 were reduced to a 20-fold and then to a
7-fold excess, respectively, with the optimal level clearly falling
somewhere between a 20- and 30-fold excess. It was gratifying
to note that for these experiments the formation of bispalmi-
toylated species 12 consistently remained at near-negligible
levels.
With the optimised conditions for the thiol-ene reaction es-
tablished, we finally reexamined the use of DMSO and DMF as
solvents for the reaction (Table 2, entries 13 and 14, respec-
tively, see Supporting Information Figures S9 and S10). Both
solvents gave monopalmitoylated product 9 selectively, al-
though only DMF (Table 2, entry 14) resulted in consumption
of peptide 10 comparable to that observed when using NMP.
The conditions optimised for this peptide were then applied
to other examples to assess the generality of the approach,
particularly on longer peptides (See Supporting Information
Figures S11–17). These results are summarised in Table 3.
In both cases, the palmitoylation process was considerably
enhanced by using our optimised conditions, which demon-
strates that complex substrates can be successfully monopalmi-
toylated. In the example of peptide 13, the conversion (81 %)
represents a considerable improvement over that previously re-
ported[4b] for this peptide (41 %). It should be noted that in the
case of peptide 13, partial oxidation of the constituent methio-
Experimental Section
General Remarks: All solvents and reagents were purchased from
commercial sources, and were used without further purification.
Fmoc-SPPS and other reactions were carried out under an air at-
mosphere without using anhydrous solvents. Aminomethyl-polysty-
rene (AM-PS) resin was synthesised “in house” as described.[21]
Fmoc-amino acids were purchased from GL Biochem (Shanghai,
China) with the following side-chain protection: Fmoc-Asn(Trt)-OH
(Trt = trityl), Fmoc-Cys(Trt)-OH, Fmoc-Cys(tBu)-OH (tBu = tert-butyl),
Fmoc-Lys(Boc)-OH (Boc = tert-butyloxycarbonyl), Fmoc-Ser(tBu)-OH,
Fmoc-Thr(tBu)-OH.
Thin-layer chromatography (TLC) was carried out using Merck silica
gel plates, and spots were visualised with UV light and/or devel-
oped using an ethanolic solution of vanillin or permanganate. Mi-
crowave irradiation was carried out with a CEM® Discover instru-
nine residues to the sulfoxides was observed, so the crude ment. NMR spectra were recorded in CDCl3 with a Bruker BRX300
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2613 © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
spectrometer operating at 300 MHz for 1H nuclei, and 75 MHz for
13C nuclei. Chemical shifts are reported in parts per million (ppm)
on the δ scale from tetramethylsilane (TMS), and were referenced
to residual solvent peaks (CDCl3: δ = 7.26 ppm for 1H NMR, δ =
(CH2), 24.8 (CH2), 22.6 (CH2), 14.0 (CH3, CH2CH3) ppm. HRMS (ESI):
calcd. for C36H51NO6SNa [M + Na]+ 648.3329; found 648.3328.
(R)-2-[(tert-Butyloxycarbonyl)amino]-3-{[2-(palmitoyloxy)ethyl]-
thio}propanoic Acid (6): Compound 3 (0.2 g, 0.90 mmol) was
treated according to General Procedure A using DMPA (0.23 g,
0.90 mmol) and vinyl palmitate (0.38 g, 1.35 mmol) in dichloro-
methane (2 mL). The crude mixture was purified by flash column
chromatography on silica gel (MeOH/CH2Cl2, 0.5:95) to give 6
1
77.0 ppm for 13C NMR). Coupling constants (J) are in Hertz (Hz). H
NMR spectroscopic data is reported as chemical shift in ppm, fol-
lowed by multiplicity and relative integral. Multiplicities are re-
ported as “s” (singlet), “br. s” (broad singlet), “d” (doublet), “dd”
(doublet of doublets), “ddd” (doublet of doublets of doublets), “dt”
(doublet of triplets), “t” (triplet), and “m” (multiplet). RP-HPLC was
carried out with a Dionex UltiMate 3000 system equipped with a
four-channel UV detector. Analytical RP-HPLC was carried out using
a Phenomenex Gemini C-18 column (5 μm; 4.6 × 150 mm) at a flow
rate of 1.0 mL/min. Semipreparative RP-HPLC was carried out using
a Foxy Jr fraction collector with a Phenomenex Gemini C18 column
(5 μm; 10.0 × 250 mm) at a flow rate of 5 mL/min, eluting using a
one-step slow gradient protocol.[22] The solvent system used was A
(0.1 % trifluoroacetic acid in H2O) and B (0.1 % trifluoroacetic acid
in acetonitrile). Peptide masses were confirmed by LC–MS using an
Agilent 1120 Compact LC system with a Hewlett Packard Series
1100 MSD mass spectrometer using ESI in the positive mode. Opti-
cal rotations were recorded with a Rudolph Autopol® IV automatic
polarimeter. Melting points were recorded with an X-4 melting-
point apparatus with a microscope. Attenuated total reflection infra-
red (ATR-IR) spectroscopy was carried out with a Bruker Alpha spec-
trometer. High-resolution mass spectra (HRMS) were obtained using
a spectrometer operating at a nominal accelerating voltage of
70 eV, or with a TOF-Q mass spectrometer.
(0.37 g, 82 %) as a yellow oil. Rf = 0.33 (MeOH/CH2Cl2, 0.5:95). [α]D23
–7.0 (c = 0.742, MeOH). IR (neat): ν = 3370.72, 2915.81, 2850.44,
=
˜
1735.03, 1687.58, 1519.59, 1471.76, 1415.78 cm–1 1H NMR
.
(300 MHz, CDCl3): δ = 9.71–8.84 (br. s, 1 H, COOH), 5.43–5.36 (m, 1
H, NH), 4.60–4.49 (m, 1 H, α-CH), 4.21 (t, J = 6.80 Hz, 2 H, SCH2CH2O),
3.14–2.93 (m, 2 H, ꢀ-CH2), 2.78 (t, J = 6.73 Hz, 2 H, SCH2CH2O), 2.30
(t, J = 7.46 Hz, 2 H, OCOCH2CH2), 1.64–1.56 (m, 2 H), 1.44 [s, 9 H,
C(CH3)3], 1.24 (s, 24 H), 0.56 (t, J = 6.82 Hz, 3 H, CH2CH3) ppm. 13C
NMR (75 MHz, CDCl3): δ = 174.7 (quat., C=O), 173.8 (quat., C=O),
155.4 (quat., OCONH), 80.5 [quat., C(CH3)3] 63.1 (CH2, SCH2CH2O),
53.2 (CH, α-CH), 34.4 (CH2, ꢀ-CH2), 34.2 (CH2, OCOCH2CH2), 31.9
(CH2), 31.2 (CH2, SCH2CH2O), 29.69 (CH2), 29.66 (CH2), 29.62 (CH2),
29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.1 (CH2), 28.2 [CH3, C(CH3)3], 28.1
(CH2), 24.9 (CH2), 22.6 (CH2), 14.1 (CH3, CH2CH3) ppm. HRMS (ESI):
calcd. for C26H49NO6SNa [M + Na]+ 526.3173; found 526.3176.
(R)-2-Acetamido-3-{[2-(palmitoyloxy)ethyl]thio}propanoic Acid
(7): Compound 4 (0.2 g, 1.22 mmol) was treated according to Gen-
eral Procedure A using DMPA (0.31 g, 1.22 mmol) and vinyl palmi-
tate (0.52 g, 1.84 mmol) in dichloromethane (2 mL). The crude mix-
ture was purified by flash column chromatography on silica gel
(MeOH/CH2Cl2, 0.5:95) to give 7 (0.48 g, 88 %) as a colourless solid,
m.p. 86.8–93.2 °C Rf = 0.08 (MeOH/CH2Cl2, 0.5:95). [α]D23 = –5.7 (c =
General Procedure A: Synthesis of S-Palmitoylated Cysteine
Building Blocks 5, 6, and 7: Vinyl palmitate (1; 1.5 equiv.) and 2,2-
dimethoxy-2-phenylacetophenone (DMPA) were added to a solu-
tion of differentially protected cysteine 2, 3, or 4 in dichloro-
methane (100 mg/1.0 mL). The reaction mixture was irradiated at a
wavelength of 365 nm using a UV lamp at room temperature. When
TLC indicated that the reaction was complete, the mixture was con-
centrated in vacuo. The residue was purified by flash column chro-
matography on silica gel to give the title compound.
0.374, MeOH). IR (neat): ν = 3328.93, 2916.77, 2850.09, 1736.25,
˜
1703.47, 1617.83, 1545.29, 1467.03, 1419.40 cm–1
.
1H NMR
(300 MHz, CDCl3): δ = 8.21–7.70 (br. s, 1 H, COOH), 6.29 (d, J =
7.51 Hz, 1 H, NH), 4.79 (ddd, J = 5.06, J = 5.49, J = 6.94 Hz, 1 H, α-
CH), 4.22 (ddd, J = 3.91, J = 6.90, J = 10.55 Hz, 2 H, SCH2CH2O), 3.12
(dd, J = 4.72, J = 14.08 Hz, 1 H, ꢀ-CH2a), 3.05 (dd, J = 5.80, J =
13.93 Hz, 1 H, ꢀ-CH2b), 2.78 (dt, J = 1.60, J = 6.55 Hz, 2 H, SCH2CH2O),
2.33 (t, J = 7.50 Hz, 2 H, OCOCH2CH2), 2.09 (s, 3 H, CH3CO), 1.65–
1.55 (m, 2 H), 1.27–1.22 (s, 24 H), 0.87 (t, J = 6.92 Hz, 3 H, CH2CH3)
ppm. 13C NMR (75 MHz, CDCl3): δ = 174.0 (quat., C=O), 172.7 (quat.,
C=O), 171.4 (quat., C=O), 62.9 (CH2, SCH2CH2O), 52.1 (CH, α-CH),
34.2 (CH2, OCOCH2CH2), 33.8 (CH2, ꢀ-CH2), 31.8 (CH2), 31.2 (CH2,
SCH2CH2O), 29.6 (CH2), 29.63 (CH2), 29.60 (CH2), 29.4 (CH2), 29.3
(CH2), 29.2 (CH2), 29.1 (CH2), 24.8 (CH2), 22.8 (CH3, CH3CO), 22.6
(CH2), 14.0 (CH3, CH2CH3) ppm. HRMS (ESI): calcd. for C23H43NO5SNa
[M + Na]+ 468.2754; found 468.2750.
(R)-2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-3-{[2-(pal-
mitoyloxy)ethyl]thio}propanoic Acid 5: Compound 2 (0.2 g,
0.58 mmol) was treated according to General Procedure A using
DMPA (0.149 g, 0.58 mmol) and vinyl palmitate (0.25 g, 0.87 mmol)
in dichloromethane (2 mL). The crude mixture was purified by flash
column chromatography on silica gel (MeOH/CH2Cl2, 0.5:95) to give
5 (0.3 g, 82 %) as a white solid, m.p. 50.3–50.7 °C. Rf = 0.33 (MeOH/
CH2Cl2, 0.5:95). [α]23 = –8.54 (c = 0.398, MeOH). IR (neat): ν =
˜
D
3318.32, 2918.39, 2850.47, 1731.80, 1693.43, 1534.48, 1466.61,
1450.17 cm–1. H NMR (300 MHz, CDCl3): δ = 7.76 (d, J = 7.6 Hz, 2
1
General Procedure for Fmoc-SPPS: Aminomethyl-polystyrene
H, ArH), 7.60 (br. s, J = 6.80 Hz, 2 H, ArH), 7.39 (t, J = 7.50 Hz, 2 H,
ArH), 7.31 (dt, J = 0.77, J = 7.50 Hz, 2 H, ArH), 6.92–6.42 (br. s, 1 H,
COOH), 5.72 (d, J = 7.66 Hz, 1 H, NH), 4.70–4.62 (m, 1 H, α-CH), 4.45–
4.38 (m, 2 H, Fmoc-CH2), 4.26–4.18 (m, 3 H, Fmoc-CH and
SCH2CH2O), 3.14 (dd, J = 4.67, J = 13.66 Hz, 1 H, ꢀ-CH2a), 3.06 (dd,
J = 5.36, J = 13.84 Hz, 1 H, ꢀ-CH2b), 2.78 (t, J = 6.40 Hz, 2 H,
SCH2CH2O), 2.29 (t, J = 7.50 Hz, 2 H, OCOCH2CH2), 1.59 (m, 2 H),
1.28–1.21 (m, 24 H), 0.88 (t, J = 6.87 Hz, 3 H, CH2CH3) ppm. 13C NMR
(75 MHz, CDCl3): δ = 174.1 (quat., C=O), 173.9 (quat., C=O), 155.9
(quat., OCONH), 144.2 (CH, ArCH), 143.69 (CH, ArCH), 143.62 (CH,
ArCH), 141.3 (CH, ArCH), 127.7 (CH, ArCH), 127.0 (CH, ArCH), 125.0
(CH, ArCH), 119.9 (CH, ArCH), 67.3 (CH2, Fmoc-CH2), 63.0 (CH2,
SCH2CH2O), 53.5 (CH, α-CH), 47.0 (CH, Fmoc-CH), 34.3 (CH2, ꢀ-CH2),
34.1 (CH2, OCOCH2CH2), 31.9 (CH2), 31.2 (CH2, SCH2CH2O), 29.67
resin (100 mg, 0.1 mmol, loading 1.0 mmol/g) was treated with
Fmoc-Val-HMPP (HMPP
=
hydroxymethylphenoxyacetic acid)
(105 mg, 0.2 mmol) and DIC (31 μL, 0.2 mmol) in a mixture of
dichloromethane and DMF (1.9:0.1 v/v; 2 mL) for 1 h at room tem-
perature. The completion of the coupling was monitored using the
Kaiser test, and if the coupling was incomplete, the coupling proce-
dure was repeated with freshly prepared reagent. Solid-phase pept-
ide synthesis was performed using a Tribute peptide synthesiser
(Protein technologies Inc.) using HATU/DIPEA for the coupling step
for 40 min at room temperature, and a solution of piperidine in
DMF (20 % v/v) for the Fmoc-deprotection step, repeated twice for
5 min at room temperature.
Resin-bound peptide 8: Target compound 8 (380 mg) was pre-
(CH2), 29.64 (CH2), 29.60 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.1 pared using the General Procedure for Fmoc-SPPS.
Eur. J. Org. Chem. 2016, 2608–2616
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2614 © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Peptide 10: Elongation of the peptide chain was achieved through
the General Procedure for Fmoc-SPPS. N-Terminal acetylation was
completed using a solution of acetic anhydride in DMF (20 % v/v)
and DIPEA (0.25 mL) for 15 min at room temperature. The resin-
bound peptide was treated with TFA/TIPS/H2O/DODT (94:1:2.5:2.5
v/v; 10 mL) for 2 h at room temperature. The TFA was evaporated
by a flow of nitrogen, then the peptide was precipitated in cold
diethyl ether, isolated by centrifugation, washed twice with cold
diethyl ether, dissolved in acetonitrile/water (1:1, v/v, containing
0.1 % TFA v/v), and lyophilised to give the crude peptide. Purifica-
tion by RP-HPLC using a semipreparative Gemini C-18 column (Phe-
nomenex, 5 μm, 10.0 × 250 mm) gave peptide 10 (74 mg, 43 %
based on a 0.1 mmol scale). MS: calcd. for [M + 2H]2+ 858.5; found
858.6.
analysed using an XTerra® MS C-18 column (Waters, 5 μm,
4.6 × 150 mm). The reaction mixture was lyophilised before methio-
nine oxide reduction. Tetrabutylammonium iodide (9 mg,
0.025 mmol) was added to the crude peptide in TFA (28 μL) at 0 °C.
After 1 min at the same temperature, the peptide was precipitated
in cold diethyl ether, isolated by centrifugation, washed twice with
cold diethyl ether, dissolved in acetonitrile/water (1:1, v/v, contain-
ing 0.1 % TFA v/v), and lyophilised to give the crude peptide. MS:
calcd. for [M + 3H]3+ 1734.2; found 1734.2.
Table 3, entry 2: Peptide 14 (0.88 mg, 0.25 μmol) was dissolved in
degassed N-methyl-2-pyrrolidone (4.7 μL), stock solution 1 (2.5 μL,
0.13 μmol) and stock solution 2 (4.94 mg, 0.018 mmol, 10 μL from
0.495 mg/μL), and then tert-butylthiol (2.3 μL, 0.02 mmol), triisopro-
pylsilane (4.1 μL, 0.02 mmol), and trifluoroacetic acid (5 % v/v;
1.4 μL) were added. The reaction mixture was irradiated at wave-
length of 365 nm using a UV lamp at room temperature, and sam-
ples were taken for LC–MS analysis at 30 min intervals. An analytical
sample was prepared by quenching with Milli-Q water, and this was
analysed using an XTerra® MS C-18 column (Waters, 5 μm,
4.6 × 150 mm). MS: calcd. for [M + 2H]2+ 1269.7; found 1269.4.
General Procedure B: Coupling of Building Blocks 5 or 7 With
Resin-Bound Peptide 8: A solution of S-palmitoylated building
block 5 (21 mg, 0.03 mmol) or 7 (15 mg, 0.03 mmol), PyBOP (17 mg,
0.03 mmol), and 2,4,6-trimethylpyridine (4.2 μL, 0.03 mmol) in DMF
(1 mL) was added to resin-bound peptide 8 (30 mg, 8 μmol based
on recovered peptidyl resin, 380 mg). The reaction mixture was left
for 1 h at room temperature, and the completion of the coupling
was monitored using the Kaiser test. If the coupling was incom-
plete, the coupling procedure was repeated with freshly prepared
reagent. The resin-bound peptide was treated with TFA/TIPS/H2O/
DODT (94:1:2.5:2.5 v/v; 1 mL) for 1 h at room temperature. The TFA
was then evaporated using a flow of nitrogen, the peptide was
precipitated in cold diethyl ether, isolated by centrifugation, washed
twice with cold diethyl ether, dissolved in acetonitrile/water (1:1,
v/v, containing 0.1 % TFA v/v), and lyophilised to give the crude
peptide.
Keywords: Synthetic methods · Click chemistry · Peptides ·
Lipids · Thiols · Cysteine
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General Procedure C: Direct Conjugation of Vinyl Palmitate (1)
and Semiprotected Peptide 10: Stock solution 1: DMPA (6.5 mg,
25.3 μmol) in degassed N-methyl-2-pyrrolidone (0.5 mL); Stock solu-
tion 2: vinyl palmitate (1) in degassed N-methyl-2-pyrrolidone (re-
quired concentration). Peptide 10 (1.71 mg, 1.0 μmol) was dissolved
in stock solution 1 (10 μL, 0.5 μmol), then tert-butylthiol, triisopro-
pylsilane, trifluoroacetic acid (5 % v/v), and stock solution 2 were
added. The reaction mixture was irradiated at wavelength of
365 nm using a UV lamp at room temperature, and samples were
taken for LC–MS analysis at 30 min intervals. An analytical sample
was prepared by quenching with Milli-Q water, and this was ana-
lysed using
a Gemini C-18 column (Phenomenex, 5 μm,
4.6 × 150 mm).
Example of General Procedure C: Table 2, entry 7: Peptide 10
(5.13 mg, 3.0 μmol) was dissolved in stock solution 1 (30 μL,
1.51 μmol), then tert-butylthiol (27 μL, 0.24 mmol), triisopropylsilane
(48.9 μL, 0.24 mmol), trifluoroacetic acid (5 % v/v; 20 μL), and stock
solution 2 (59.1 mg, 0.21 mmol, 300 μL from 0.2 mg/μL) were
added. Upon completion of the reaction, as monitored by RP-HPLC,
the crude material was directly purified by RP-HPLC using a semi-
preparative diphenyl column (Vydac, 5 μm, 10.0 × 250 mm) to give
peptide 9 (4.29 mg, 72 %). MS: calcd. for [M + 2H]2+ 999.5; found
999.9.
[8] J. Kopycinski, M. Osman, P. D. Griffiths, V. C. Emery, J. Med. Virol. 2010,
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2012, 109, 261–266.
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Table 3, entry 1: Peptide 13 (1.23 mg, 0.25 μmol) was dissolved in
degassed N-methyl-2-pyrrolidone (4.7 μL), stock solution 1 (2.5 μL,
0.13 μmol), and stock solution 2 (4.94 mg, 0.018 mmol, 10 μL from
0.495 mg/μL), and then tert-butylthiol (2.3 μL, 0.02 mmol), triisopro-
pylsilane (4.1 μL, 0.02 mmol), and trifluoroacetic acid (5 % v/v;
1.4 μL) were added. The reaction mixture was irradiated at wave-
length of 365 nm using a UV lamp at room temperature, and sam-
ples were taken for LC–MS analysis at 30 min intervals. An analytical
sample was prepared by quenching with Milli-Q water, and this was
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Full Paper
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Received: November 29, 2015
Published Online: March 29, 2016
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim