A trimethyllysine-containing trityl tag for solubilizing hydrophobic peptides

Article abstract of DOI:10.1039/c9ob02253h

Hydrophobic membrane peptides/proteins having low water solubility are often difficult to prepare. To overcome this issue, temporal introduction of solubilizing tags has been demonstrated to be beneficial. Following our recent work on the solubilization of a difficult target by using a hydrophilic oligo-Lys tag bearing a trityl linker (Trt-K method), this paper describes a comparative study of the solubilizing abilities of several peptidic trityl tags containing Lys, Arg, Glu, Asn, N^?-tri-Me-Lys or Cys-sulfonate using two hydrophobic model peptides. Among the tags evaluated, that containing N^?-tri-Me-Lys exhibits superior solubilizing ability.

Full text of DOI:10.1039/c9ob02253h

Organic &
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PAPER
A trimethyllysine-containing trityl tag for
solubilizing hydrophobic peptides†
Cite this: Org. Biomol. Chem., 2019,
17, 10228
Shun Masuda, Shugo Tsuda
and Taku Yoshiya
*
Hydrophobic membrane peptides/proteins having low water solubility are often difficult to prepare. To over-
come this issue, temporal introduction of solubilizing tags has been demonstrated to be beneficial. Following
our recent work on the solubilization of a difficult target by using a hydrophilic oligo-Lys tag bearing a trityl
linker (Trt-K method), this paper describes a comparative study of the solubilizing abilities of several peptidic
trityl tags containing Lys, Arg, Glu, Asn, Nε-tri-Me-Lys or Cys-sulfonate using two hydrophobic model
peptides. Among the tags evaluated, that containing Nε-tri-Me-Lys exhibits superior solubilizing ability.
Received 17th October 2019,
Accepted 20th November 2019
DOI: 10.1039/c9ob02253h
rsc.li/obc
terized by the following features: (1) an introducing reagent for
the trityl alcohol-type tag, “Trt(OH)-Kn”, can be readily pre-
Introduction
Chemical peptide/protein synthesis mainly consists of two pared using commercially available 4-(diphenylhydroxymethyl)
steps: peptide chain construction and purification using high benzoic acid; (2) the tag can be attached to the thiol group in
performance liquid chromatography (HPLC). The first step is the unprotected peptide by simply adding the introducing
well established and based on methods such as solid-phase reagent under acidic conditions such as neat 1,1,1,3,3,3-hexa-
peptide synthesis (SPPS),1 native chemical ligation (NCL),2 and fluoroisopropanol (HFIP), which can dissolve hydrophobic and
related techniques.3 In contrast, there is still room for improve- practically insoluble peptides,8 or an acidified thiol-additive-
ment in the second step, since hydrophobic membrane pep- free NCL reaction mixture in a one-pot manner; (3) the tag can
tides/proteins having low water solubility are difficult to purify be quickly and cleanly detached by a standard deprotection
by HPLC.4 Additionally, the low solubility of these compounds approach using trifluoroacetic acid (TFA) with a cation scaven-
also hampers further development of NCL processes in which ger such as triisopropylsilane (TIS). Unlike the other known
unprotected peptide segments are ligated in an aqueous solubilizing tag systems,6 the solubilizing tag in our method
solvent.5 To solve this problem, many researchers developed can be attached “on demand”, without the need for starting a
solubilizing tags such as a phenylacetamidomethyl linker new preparation when the target peptide segment is found in-
method,6g a removable backbone modification method,6h and soluble. This is a great advantage because it is generally
a canaline tag.6m In this context, we recently developed a difficult to estimate the solubility of each protein intermediate
simple solubilizing trityl-type tag system (Trt-K, where Trt and prior to the synthesis. To broaden the scope of our method
K denote trityl and Lys, respectively) (Fig. 1),7 which is charac- and facilitate its further application to challenging targets, we
herein report a comparative study of the solubilizing abilities
of ionic/nonionic peptide tags having different structures. We
compare several peptide tags composed of basic residues (Lys/
Arg), an acidic residue (Glu), or a nonionic residue (Asn).
Furthermore, peptide tags composed of Nε-tri-Me-Lys or Cys-
sulfonate as pH-independent analogs are evaluated. As a
result, the Nε-tri-Me-Lys-containing tag is demonstrated to
exhibit the best solubilizing ability in solution.
Fig. 1 Solubilizing trityl-type tag system.
Results
Peptide Institute, Inc., Ibaraki, Osaka 567-0085, Japan.
E-mail: t.yoshiya@peptide.co.jp; https://www.peptide.co.jp/en
†Electronic supplementary information (ESI) available: Solubility of tagged SPP4
in different solvents for various ligation techniques, and characterization data
such as NMR spectra, HPLC traces, and MS spectra. See DOI: 10.1039/
c9ob02253h
Preparation of tag-introducing reagents
To compare the solubilizing abilities of several Trt-X tags, we
designed several examples: Trt-Kn, where K denotes Lys and
n = 10, 8, 5, 4, 3, branched 8;9 Trt-Rn, where R denotes Arg and
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Scheme 1 Synthesis of Trt-X tags and structures of selected solubilizing tags.
n = 8, 5, 3; Trt-E5, where E denotes Glu; Trt-N5, where N
denotes Asn;10 Trt-[K(Me3)]5, where K(Me3) denotes Nε-tri-Me-
Lys; and Trt-[C(O3H)]5, where C(O3H) denotes Cys-sulfonate.
Each tag-introducing reagent was prepared straightforwardly
by a standard Fmoc SPPS using Wang resin (Scheme 1). After
final acylation using commercially available 4-(diphenylhydrox-
ymethyl)benzoic acid, TFA treatment without a cation scaven-
ger such as TIS followed by RP-HPLC purification afforded
each desired trityl alcohol-type tag-introducing reagent
successfully.
Solubilization of Ac-Val-Val-Cys-Val-Val-NH2
Having several tag-introducing reagents in hand, we pro-
ceeded with their attachment under standard HFIP con-
ditions to the first solubilizing target Ac-Val-Val-Cys-Val-Val-
NH2 (1), which is a highly hydrophobic model peptide.11 The
different tags were directly attached to crude 1 after Fmoc
SPPS using Rink amide resin, and the respective tagged pep-
tides were purified by RP-HPLC. The resulting HPLC profiles
are summarized in Fig. 2. As we expected, the longer Lys/Arg-
tagged peptide was eluted faster than its shorter counterpart.
Moreover, the branched tagged peptide (branched K8) was
eluted later than the linear derivative. The sulfonate-contain-
Fig. 2 HPLC profiles of tagged Ac-VVCVV-NH2. HPLC conditions:
column, YMC-Pack ODS-A (4.6 × 150 mm); elution, 10%–60% CH3CN in
ing tagged peptide12 was eluted very fast because it remains 0.1% TFA (25 min) at 40 °C; flow rate, 1.0 mL min−1; detection, 220 nm.
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Fig. 4 HPLC profiles of tagged SPP4. HPLC conditions: column,
YMC-Pack ODS-A (4.6 × 150 mm); elution, 10%–98% CH3CN in 0.1%
TFA (25 min) at 40 °C; flow rate, 1.0 mL min−1; detection, 220 nm.
tagged 2 because its purification by RP-HPLC was difficult.12
As shown in Fig. 4, the elution profile obtained by HPLC was
similar to that of 1. Thus, the longer Trt-K/R-tagged peptides
eluted faster than the shorter Trt-K/R-tagged peptides, the
branched tagged peptide eluted later than the linear one, and
the Trt-K(Me3)-tagged peptide eluted later than the corres-
ponding nonmethylated derivative. With respect to the solubil-
ization ability (Fig. 5), a similar tendency to that of peptide 1
was observed. The Trt-K/R-tagged peptides were found to dis-
solve well in solutions of low pH but not under neutral con-
ditions, and the longer Trt-K/R tags solubilized 2 more than
their shorter counterparts. Furthermore, the Trt-K tags showed
better solubilizing ability than the Trt-R tags. Additionally, the
Trt-[K(Me3)]5-tagged peptide was more soluble than the Trt-K5/
R5-tagged peptides.
Fig. 3 Solubilization ability of tagged Ac-VVCVV-NH2. Ac-VVCVV-NH2
(1) was not soluble (0.00 mM) without a tag. a Gel was formed to prevent
solubility evaluation.
in anionic form under 0.1% TFA aq conditions and it does
not form TFA salt during HPLC. In contrast, the Glu-contain-
ing peptide was eluted very late, since it is not anionic under
the HPLC conditions. The Asn-containing peptide was also
eluted late. With respect to the solubilization ability under
acidic conditions (Fig. 3), all basic tags [K, R, K(Me3)] and the
strongly acidic tag C(O3H) solubilized 1 well. In contrast, the
weakly acidic tag E and the nonionic tag N did not solubilize
1 efficiently under these acidic conditions. Under neutral
conditions, we observed a dependence of the solubilizing
ability on the peptide length of linear Kn and Rn, and linear
K8 solubilized 1 better than branched K8. The Kn tags exhibi-
ted higher solubilizing ability than the corresponding Rn
tags.13 Furthermore, the [K(Me3)]5 tag solubilized 1 more
than the corresponding K5/R5 tags. Additionally, we observed
that both strongly and weakly acidic tags [C(O3H) and E] solu-
bilized 1 well, whereas the nonionic tag N did not work under
neutral conditions.
Discussion
In this paper, we compare the solubilizing abilities of a series
of Trt-based tags, i.e., Trt-K/R/K(Me3)/C(O3H)/E/N, which is
expected to depend on the electrical properties derived from
their different pKa values.15 Accordingly, basic tags [Trt-K/R/K
(Me3)] are cationic under both neutral and acidic conditions;
the strongly acidic tag Trt-C(O3H) is anionic under both
neutral and cationic conditions; the weakly acidic tag Trt-E is
Solubilization of SPP4
Next, we evaluated the solubilizing ability using as a second anionic under neutral conditions but not under acidic con-
target peptide SPP4 (2), which is a known hydrophobic peptide ditions; and the neutral tag Trt-N is nonionic regardless of the
found in membrane proteins.6d,14 In a similar manner to that solution pH. The results obtained for the retention times and
described for model peptide 1, several basic tags were attached solubilization abilities of the two hydrophobic peptides evalu-
to crude 2, and the tagged series 2 was purified by RP-HPLC. ated in this study are basically in agreement with that theory;
In this case, we could not prepare a sulfonate-containing longer/ionized tags showed good solubilization ability com-
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Fig. 5 Solubilization ability of tagged SPP4. SPP4 (2) was not soluble (0.00 mM) without a tag. a Not tested.
pared with shorter/nonionized tags. However, to our surprise,
the Trt-K/R tag did not work well in solution under neutral
conditions. In fact, the Trt-K(Me3) tag showed higher solubil-
ization ability than the Trt-K/R tags having the same length
under neutral conditions. These results cannot be explained
simply by the pKa theory. On the basis of the pKa of Lys and
Arg, the Trt-K/R tags would be protonated consistently under
neutral and acidic conditions, and should have similar solubil-
ization abilities regardless of the solution pH. This contradic-
tion can be rationalized in terms of the crowd effect (Fig. 6);
under crowded conditions such as those found in the Trt-X
tags, the interaction between functionalities would affect the
protonation state. Similar protonation inhibitions are well
known in peptide chemistry as a neighboring effect; for
example, N-terminal Asn(Trt) deprotection is slow during final
TFA treatment in Fmoc SPPS,16 and Nα-Boc deprotection from
N-terminal Boc-His(Bom) on the resin is slow during Boc
Fig. 6 Proposed structures of tags in the protonation state.
SPPS.17 In these cases, a protonated base prevents the protona-
tion of a neighboring base, which is essential for the reaction
progress. In our case, especially under neutral conditions, the
crowd effect would decrease the solubilizing ability of Trt-K/R. tively weaker basicity than ε-amino groups are present on the
In addition, the side-chain guanidine groups of Arg would surface, and their protonation would be inhibited by the
tend to aggregate via π–π stacking under neutral conditions,18 crowd effect. Although the anionic Trt-C(O3H) tag has good
thereby mitigating the solubilizing ability. Such stacked struc- solubilizing ability, its purification is difficult. The Trt-E tag
tures have been crystallographically observed.19 This phenom- does not solubilize the peptide under acidic conditions, and
enon can also be invoked to explain the lower solubilizing the Trt-N tag has no obvious solubilizing ability. From these
ability of the branched K8 tag compared to that of linear K8. In results, it can be concluded that Trt-K(Me3) is the best tag
the case of the branched tag, α-amino groups possessing rela- among those evaluated in this study.
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Experimental section
Conclusions
Synthesis of Fmoc-amino acid and peptides
In this study, we compared the solubilizing abilities of several
Trt-X tags using two hydrophobic peptides. The results
obtained indicate that the Trt-K(Me3) tag is superior with
respect to solubilizing ability among the tags examined.
Additionally, the sulfonate-containing tag Trt-C(O3H) solubil-
izes the hydrophobic peptide well; however, its HPLC purifi-
cation is difficult. These results contribute to further develop-
ment of chemical hydrophobic peptide/protein preparation.
Fmoc-Cys(O3H)-OH. To a solution of cysteic acid (1.0 g,
5.91 mmol) and Na2CO3 (0.626 g, 5.91 mmol) in H2O (25 mL)
was added Fmoc-OSu solution (2.39 g, 7.09 mmol in 25 mL
acetone). The reaction mixture was stirred at room temperature
for 2 h, and then acetone was removed under reduced
pressure. The aqueous residue was washed with AcOEt/hexane
(v/v, 1/1). The aqueous layer was acidified with 1 M HCl to pH
2 and then subjected to preparative HPLC to yield the title
1
compound (1.57 g, 80%). H NMR (400 MHz, DMSO-d6) δ 2.86
(dd, J = 13.7 and 5.0 Hz, 1H), 2.90 (dd, J = 13.7 and 6.4 Hz,
1H), 4.10–4.33 (m, 4H), 7.28–7.37 (m, 2H), 7.42 (t, J = 7.3 Hz,
2H), 7.47 (d, J = 6.9 Hz, 1H) 7.70 (dd, J = 7.3 and 2.7 Hz, 2H),
7.89 (d, J = 7.3 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 46.7,
51.0, 51.4, 65.9, 120.2, 125.3, 125.4, 127.2, 127.7, 140.7, 140.8,
143.8, 143.9, 155.7, 172.3; LRMS (M − H) calcd for C18H16NO7S
390.1, found 390.1.
Ac-VVCVV-NH2 (1). The peptide was assembled on Rink
amide resin (0.40 mmol) using an automated Fmoc SPPS pro-
cedure as described in General information (Fmoc-Xaa: 2.5
equiv.). The subsequent deprotection of the resin was carried
out with TFA/TIS/H2O (v/v, 95/2.5/2.5) for 1.5 h to give a crude
product. The peptide was used to the next reaction without
further purification. Analytical HPLC: tR = 14.5 min (1–60%
CH3CN/0.1% TFA for 25 min); LRMS (M + H) calcd for
C25H47N6O6S 559.3, found 559.3.
STGCILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4 (2). The
peptide was assembled on Rink amide resin (0.25 mmol)
using the automated Fmoc SPPS procedure as described in
General information (Fmoc-Xaa and Fmoc-Val-Thr(ΨMe,Mepro):
4 equiv.). The subsequent deprotection of the resin was carried
out with TFA/TIS/H2O/DMB (v/v, 92.5/2.5/2.5/2.5) for 1.5 h to
give a crude product. The peptide was used for the next reac-
Experimental
General information
Materials. All reagents and solvents were obtained from
Peptide Institute, Inc. (Osaka, Japan), FUJIFILM Wako Pure
Chemical Corporation (Osaka, Japan), Tokyo Chemical
Industry Co., Ltd (Tokyo, Japan), Nacalai Tesque, Inc. (Kyoto,
Japan), Watanabe Chemical Industries, Ltd (Hiroshima,
Japan), Merck KGaA (Darmstadt, Germany) and Sigma-Aldrich
Co. LLC (St Louis, MO).
HPLC, MS and NMR. Preparative HPLC was carried out on a
Shimadzu liquid chromatograph Model LC-8A (Kyoto, Japan)
with a YMC-Pack ODS-A (30 × 250 mm) or a YMC-Actus Triart
C18 (30 × 250 mm) and the following solvent systems: 0.1%
TFA in H2O and 0.1% TFA in CH3CN or 0.1 M NH4OAc buffer
(pH 7.0) and 60% CH3CN/0.1 M NH4OAc buffer (pH 7.0) at a
flow rate of 20 mL min−1 with detection at 220 nm. Analytical
HPLC was performed on a Shimadzu liquid chromatograph
Model LC-10A (Kyoto, Japan) with a YMC-Pack ODS-A (4.6 ×
150 mm) or a YMC-Triart C18 (4.6 × 150 mm) and the follow-
ing solvent systems: 0.1% TFA in H2O and 0.1% TFA in CH3CN
or 0.1 M NH4OAc buffer (pH 7.0) and 60% CH3CN/0.1 M
NH4OAc buffer (pH 7.0) at a flow rate of 1 mL min−1 (40 °C)
with detection at 220 nm. Low resolution mass spectra (LRMS)
were observed with an Agilent G1956B LC/MSD detector using
an Agilent 1100 series HPLC system; for deconvolution, the
observed masses (most abundant masses) were derived from
the experimental m/z values for each protonation state of a
target peptide. 1H-/13C-NMR spectra were recorded on a
JEOL-ECX400 spectrometer (Tokyo, Japan), as solutions in
deuterated solvents as specified. Chemical shift values (δ) are
given in parts per million (ppm) using the residual solvent as
the internal standard.
SPPS. Automated Fmoc SPPS was performed on an
ABI 433A peptide synthesizer (Applied Biosystems, USA).
The peptide chains except Trt(OH)-[K(Me3)]5 and Trt(OH)-
[C(O3H)]5 were elongated using the coupling protocol of
Fmoc-amino acid/DIC/OxymaPure.20 Trt(OH)-[K(Me3)]5 and
Trt(OH)-[C(O3H)]5 were manually elongated using the coupl-
ing protocol of HCTU/DIEA.21 The following side-chain-
protected amino acids and pseudoproline unit were employed:
Arg(Pbf), Asn(Trt), Asp(OtBu), Glu(OtBu), Ser(tBu), Cys(Trt),
Lys(Boc), Lys(Fmoc), Ser(tBu), Thr(tBu), Trp(Boc), Tyr(tBu), and
Val-Thr(ΨMe,Mepro).
tion without further purification. Analytical HPLC: tR
=
22.3 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
calcd for C153H234N34O39S2 3236.7, found 3237.6.
Trt(OH)-K10. The peptide was assembled on Fmoc-Lys(Boc)-
Wang resin (0.40 mmol) using the automated Fmoc SPPS pro-
cedure as described in General information (Fmoc-Lys(Boc):
2.5 equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.).
The subsequent deprotection of the resin was carried out with
TFA/H2O (v/v, 95/5) for 30 min to give a crude product, which
was purified by preparative HPLC to yield the title compound
(641 mg, 59%). Analytical HPLC: tR = 14.4 min (1–40% CH3CN/
0.1% TFA for 25 min); LRMS (M + H) calcd for C80H137N20O13
1586.1, found 1586.0.
Trt(OH)-K8. The peptide was assembled on Fmoc-Lys(Boc)-
Wang resin (0.25 mmol) using the automated Fmoc SPPS pro-
cedure as described in General information (Fmoc-Xaa: 4
equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.).
The subsequent deprotection of the resin was carried out with
TFA/H2O (v/v, 95/5) for 40 min to give a crude product, which
was purified by preparative HPLC to yield the title compound
(361 mg, 64%). Analytical HPLC: tR = 14.8 min (1–40% CH3CN/
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0.1% TFA for 25 min); LRMS (M + H) calcd for C68H113N16O11 The subsequent deprotection of the resin was carried out with
1329.9, found 1329.8. TFA/H2O/thioanisole (v/v/v, 95/2.5/2.5) for 25 min to give a
Trt(OH)-K5. The peptide was assembled on Fmoc-Lys(Boc)- crude product, which was purified by preparative HPLC to
Wang resin (0.40 mmol) using the automated Fmoc SPPS pro- yield the title compound (265 mg, 59%). Analytical HPLC: tR
=
cedure as described in General information (Fmoc-Lys(Boc): 18.8 min (1–40% CH3CN/0.1% TFA for 25 min); LRMS (M + H)
2.5 equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.). calcd for C38H53N12O6 773.4, found 773.4.
The subsequent deprotection of the resin was carried out with
Trt(OH)-E5. The peptide was assembled on the Fmoc-Glu
TFA/H2O (v/v, 95/5) for 40 min to give a crude product, which (OtBu)-Wang resin (0.25 mmol) using the automated Fmoc
was purified by preparative HPLC to yield the title compound SPPS procedure as described in General information (Fmoc-
(395 mg, 65%). Analytical HPLC: tR = 15.7 min (1–40% CH3CN/ Xaa: 4 equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5
0.1% TFA for 25 min); LRMS (M + H) calcd for C50H77N10O8 equiv.). The subsequent deprotection of the resin was carried
945.6, found 945.5.
out with TFA/H2O (v/v, 95/5) for 1 h to give a crude product,
Trt(OH)-K4. The peptide was assembled on Fmoc-Lys(Boc)- which was purified by preparative HPLC to yield the title com-
Wang resin (0.40 mmol) using the automated Fmoc SPPS pro- pound (144 mg, 61%). Analytical HPLC: tR = 14.3 min (10–60%
cedure as described in General information (Fmoc-Xaa: 2.5 CH3CN/0.1% TFA for 25 min); LRMS (M − H) calcd for
equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.). C45H50N5O18 948.3, found 948.2.
The subsequent deprotection of the resin was carried out with
Trt(OH)-N5. The peptide was assembled on Rink amide resin
TFA/H2O (v/v, 95/5) for 1 h to give a crude product, which was (0.4 mmol) using the automated Fmoc SPPS procedure as
purified by preparative HPLC to yield the title compound described in General information (Fmoc-Xaa: 2.5 equiv., Fmoc-
(328 mg, 64%). Analytical HPLC: tR = 16.3 min (1–40% CH3CN/ Asp-OtBu is used as C-terminal Xaa, 4-(diphenylhydroxy-
0.1% TFA for 25 min); LRMS (M + H) calcd for C44H65N8O7 methyl)benzoic acid: 2.5 equiv.). The subsequent deprotection
817.5, found 817.4.
of the resin was carried out with TFA/H2O (v/v, 95/5) for 1 h to
Trt(OH)-K3. The peptide was assembled on Fmoc-Lys(Boc)- give a crude product, which was purified by preparative HPLC
Wang resin (0.40 mmol) using the automated Fmoc SPPS pro- to yield the title compound (234 mg, 67%). Analytical HPLC: tR
cedure as described in General information (Fmoc-Xaa: 2.5 = 12.9 min (10–60% CH3CN/0.1% TFA for 25 min); LRMS (M −
equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.). H) calcd for C40H45N10O13 873.3, found 873.3.
The subsequent deprotection of the resin was carried out with
Trt(OH)-branched K8. The peptide was assembled on the
TFA/H2O (v/v, 95/5) for 40 min to give a crude product, which Fmoc-Lys(Aloc)-Wang resin (0.25 mmol) using a manual Fmoc
was purified by preparative HPLC to yield the title compound SPPS procedure (Fmoc-Lys(Fmoc): 4 equiv. to amino group)
(202 mg, 49%). Analytical HPLC: tR = 17.4 min (1–40% CH3CN/ without final Fmoc deprotection. The resin was treated with
0.1% TFA for 25 min); LRMS (M + H) calcd for C38H53N6O6 Pd(PPh3)4 (72.2 mg, 0.0625 mmol) and PhSiH3 (1.53 mL,
689.4, found 689.4.
12.5 mmol) in CH2Cl2 for 1 h. Then, 4-(diphenylhydroxy-
Trt(OH)-R8. The peptide was assembled on the Fmoc-Arg methyl)benzoic acid was coupled using the DIC/Oxyma
(Pbf)-Wang resin (0.40 mmol) using the automated Fmoc SPPS method, followed by deprotection of the Fmoc group with 20%
procedure as described in General information (Fmoc-Xaa: 2.5 piperidine/DMF. The obtained resin was treated with TFA/H2O
equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.). (v/v, 95/5) for 1 h to give a crude product, which was purified
The subsequent deprotection of the resin was carried out with by preparative HPLC to yield the title compound (77.8 mg,
TFA/H2O/thioanisole (v/v/v, 92.5/2.5/5) for 25 min to give a 14%). Analytical HPLC: tR = 15.9 min (1–40% CH3CN/0.1%
crude product, which was purified by preparative HPLC to TFA for 25 min); LRMS (M + H) calcd for C68H113N16O11
yield the title compound (202 mg, 20%). Analytical HPLC: tR
=
1329.9, found 1329.8.
16.8 min (1–40% CH3CN/0.1% TFA for 25 min); LRMS (M + H)
calcd for C68H113N32O11 1553.9, found 1553.9.
Trt(OH)-[K(Me3)]5. The peptide was assembled on the Fmoc-
Gly-Wang resin (0.1 mmol) using the manual Fmoc SPPS pro-
Trt(OH)-R5. The peptide was assembled on the Fmoc-Arg cedure (Fmoc-Lys(Me3)·Cl: 2 equiv., 4-(diphenylhydroxymethyl)
(Pbf)-Wang resin (0.40 mmol) using the automated Fmoc SPPS benzoic acid: 2 equiv.). The subsequent deprotection of the
procedure as described in General information (Fmoc-Xaa: 2.5 resin was carried out with TFA/H2O (v/v, 95/5) for 1 h to give a
equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.). crude product, which was purified by preparative HPLC to
The subsequent deprotection of the resin was carried out with yield the title compound (72.6 mg, 41%). Analytical HPLC: tR =
TFA/H2O/thioanisole (v/v/v, 95/2.5/2.5) for 25 min to give a 12.1 min (10–60% CH3CN/0.1% TFA for 25 min); LRMS (M)
crude product, which was purified by preparative HPLC to calcd for C67H114N11O95+ 243.4, found 243.5.
yield the title compound (248 mg, 37%). Analytical HPLC: tR
17.4 min (1–40% CH3CN/0.1% TFA for 25 min); LRMS (M + H) Gly-Wang resin (0.25 mmol) using the manual Fmoc SPPS pro-
calcd for C50H77N20O8 1085.6, found 1085.5. cedure (Fmoc-Cys(O3H): 2 equiv., 4-(diphenylhydroxymethyl)
=
Trt(OH)-[C(O3H)]5. The peptide was assembled on the Fmoc-
Trt(OH)-R3. The peptide was assembled on the Fmoc-Arg benzoic acid: 2 equiv.). The subsequent deprotection of the
(Pbf)-Wang resin (0.40 mmol) using the automated Fmoc SPPS resin was carried out with TFA/H2O (v/v, 95/5) for 1 h to give a
procedure as described in General information (Fmoc-Xaa: 2.5 crude product, which was purified by preparative HPLC to
equiv., 4-(diphenylhydroxymethyl)benzoic acid: 2.5 equiv.). yield the title compound (96.9 mg, 35%). Analytical HPLC: tR =
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9.2 min (1–60% CH3CN/0.1% TFA for 25 min); LRMS (M − H) peak (8.8 mg, 35% isolated yield). Analytical HPLC: tR
=
calcd for C37H43N6O24S5 1115.1, found 1115.0.
14.2 min (10–60% CH3CN/0.1 M NH4OAc buffer (pH 7) for
Attachment of Trt(OH)-X to Ac-VVCVV-NH2
25 min); LRMS (M − H) calcd for C62H87N12O29S6 1655.4,
General procedure. Ac-VVCVV-NH2 (1 equiv., 10 mM) and Trt found 1655.3.
(OH)-X (1.1 equiv.) were dissolved in HFIP.6 After stirring for
1 h at room temperature, the reaction mixture was concen-
trated and subjected to preparative HPLC.
Attachment of Trt(OH)-X to SPP4
General procedure. SPP4 (1 equiv., 10 mM) and Trt(OH)-X
(1.1 equiv.) were dissolved in HFIP or TFA. After stirring for 1 h
Ac-VVC(Trt-K10)VV-NH2. 27.8 mg, 85% isolated yield. at room temperature, the reaction mixture was concentrated
Analytical HPLC: tR = 12.9 min (10–60% CH3CN/0.1% TFA for and subjected to preparative HPLC.
25 min); LRMS (M + H) calcd for C105H181N26O18S 2126.4,
found 2126.3.
STGC(Trt-K10)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-K10. 46.1 mg, 35% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-K8)VV-NH2. 25.6 mg, 92% isolated yield. 17.7 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 13.2 min (10–60% CH3CN/0.1% TFA for calcd for C233H368N54O51S2 4804.7, found 4805.4.
25 min); LRMS (M + H) calcd for C93H157N22O16S 1870.2,
found 1870.1.
STGC(Trt-K8)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-K8. 37.3 mg, 33% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-branched K8)VV-NH2. 25.1 mg, 90% isolated yield. 18.0 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 13.5 min (10–60% CH3CN/0.1% TFA for calcd for C221H344N50O49S2 4548.5, found 4549.0.
25 min); LRMS (M + H) calcd for C93H157N22O16S 1870.2,
found 1870.1.
STGC(Trt-branched K8)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2
= SPP4-Trt-branched K8. 31.6 mg, 34% isolated yield. Analytical
Ac-VVC(Trt-K5)VV-NH2. 26.1 mg, 85% isolated yield. HPLC: tR = 18.0 min (10–98% CH3CN/0.1% TFA for 25 min);
Analytical HPLC: tR = 14.0 min (10–60% CH3CN/0.1% TFA for LRMS (ESI) calcd for C221H344N50O49S2 4548.5, found 4549.1.
25 min); LRMS (M + H) calcd for C75H121N16O13S 1485.9,
found 1485.8.
STGC(Trt-K5)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-K5. 27.9 mg, 28% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-K4)VV-NH2. 24.0 mg, 88% isolated yield. 18.6 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 14.5 min (10–60% CH3CN/0.1% TFA for calcd for C203H308N44O46S2 4164.3, found 4164.5.
25 min); LRMS (M + H) calcd for C69H109N14O12S 1357.8,
found 1357.8.
STGC(Trt-K4)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-K4. 12.8 mg, 14% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-K3)VV-NH2. 26.3 mg, 84% isolated yield. 19.0 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 15.2 min (10–60% CH3CN/0.1% TFA for calcd for C197H296N42O45S2 4036.2, found 4036.6.
25 min); LRMS (M + H) calcd for C63H97N12O11S 1229.7, found
1229.6.
STGC(Trt-K3)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-K3. 15.8 mg, 18% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-R8)VV-NH2. 22.1 mg, 73% isolated yield. 19.6 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 14.1 min (10–60% CH3CN/0.1% TFA for calcd for C191H284N40O44S2 3908.1, found 3908.1.
25 min); LRMS (M + H) calcd for C93H157N38O16S 2094.2,
found 2094.2.
STGC(Trt-R8)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-R8. 32.8 mg, 28% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-R5)VV-NH2. 29.1 mg, 88% isolated yield. 18.1 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 14.8 min (10–60% CH3CN/0.1% TFA for calcd for C221H344N66O49S2 4772.6, found 4773.2.
25 min); LRMS (M + H) calcd for C75H121N26O13S 1625.9,
found 1625.9.
STGC(Trt-R5)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-R5. 27.5 mg, 27% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-R3)VV-NH2. 27.6 mg, 83% isolated yield. 18.7 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 15.9 min (10–60% CH3CN/0.1% TFA for calcd for C203H308N54O46S2 4304.3, found 4304.6.
25 min); LRMS (M + H) calcd for C63H97N18O11S 1313.7, found
1313.7.
STGC(Trt-R3)ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2 = SPP4-
Trt-R3. 15.9 mg, 17% isolated yield. Analytical HPLC: tR
=
Ac-VVC(Trt-E5)VV-NH2. 16.5 mg, 74% isolated yield. 19.6 min (10–98% CH3CN/0.1% TFA for 25 min); LRMS (ESI)
Analytical HPLC: tR = 17.2 min (10–60% CH3CN/0.1% TFA for calcd for C191H284N46O44S2 3992.1, found 3992.2.
25 min); LRMS (M − H) calcd for C70H94N11O23S 1488.6, found
STGC{Trt-[K(Me3)]5}ILLGGLFIYDVFWVFGTNVMVTVAKS-NH2
=
1488.5.
SPP4-Trt-K[(Me3)]5. 11.5 mg, 13% isolated yield. Analytical
Ac-VVC(Trt-N5)VV-NH2. 7.1 mg, 70% isolated yield. Analytical HPLC: tR = 19.9 min (10–98% CH3CN/0.1% TFA for 25 min);
HPLC: tR = 16.7 min (10–60% CH3CN/0.1% TFA for 25 min); LRMS (M) calcd for C220H346N45O47S25+ 887.3, found 887.3.
LRMS (M + H) calcd for C65H91N16O18S 1415.6, found 1415.6.
Evaluation of the solubility of Trt-X tagged peptides. The
Ac-VVC{Trt-[K(Me3)]5}VV-NH2. 21.3 mg, 81% isolated yield. solubility of Trt-X tagged peptides was determined by calcu-
Analytical HPLC: tR = 15.5 min (10–60% CH3CN/0.1% TFA for lation of peak areas of standard solutions and the supernatant
25 min); LRMS (M) calcd for C92H158N17O14S5+ 351.4, found of saturated solutions using analytical HPLC. The standard
351.6.
solutions were prepared by dissolving the peptides at 1 mg
Ac-VVC{Trt-[C(O3H)]5}VV-NH2. The title peptide was isolated mL−1 in DMSO. The saturated solutions were prepared by
using a 0.1 M NH4OAc buffer (pH 7) solvent system as a single adding small aliquots of buffers into the peptides. The satu-
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rated solutions were centrifuged and the supernatants were
diluted with 50% AcOH/H2O or 3 M Gn·HCl in 50% AcOH/
H2O, respectively. The standard solutions and the diluted
supernatant solutions were compared by peak areas using
analytical HPLC.
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Conflicts of interest
There are no conflicts to declare.
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under aqueous conditions without denaturing agents such
as guanidine·HCl. Incidentally, to evaluate the applicability
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of our Trt-K/R method for NCL, α-ketoacid–hydroxylamine 20 R. Subirós-Funosas, R. Prohens, R. Barbas, A. El-Faham
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