1,2-Dimethylindole-3-sulfonyl (MIS) as protecting group for the side chain of arginine

Article abstract of DOI:10.1039/b904836g

The protection of arginine (Arg) side chains is a crucial issue in peptide chemistry because of the propensity of the basic guanidinium group to produce side reactions. Currently, sulfonyl-type protecting groups, such as 2,2,5,7,8-pentamethylchroman (Pmc)

Full text of DOI:10.1039/b904836g

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1,2-Dimethylindole-3-sulfonyl (MIS) as protecting group for the side
chain of arginine†
a
b
b
a,c,d
and Fernando Albericio*a,c,e
´
Albert Isidro, Daniel Latassa, Matthieu Giraud, Mercedes Alvarez*
Received 9th March 2009, Accepted 16th March 2009
First published as an Advance Article on the web 23rd April 2009
DOI: 10.1039/b904836g
The protection of arginine (Arg) side chains is a crucial issue in peptide chemistry because of the
propensity of the basic guanidinium group to produce side reactions. Currently, sulfonyl-type protecting
groups, such as 2,2,5,7,8-pentamethylchroman (Pmc) and 2,2,4,6,7-pentamethyldihydrobenzofurane
(Pbf), are the most widely used for this purpose. Nevertheless, Arg side chain protection remains
problematic as a result of the acid stability of these two compounds. This issue is even more relevant in
Arg-rich sequences, acid-sensitive peptides and large-scale syntheses. The 1,2-dimethylindole-3-sulfonyl
(MIS) group is more acid-labile than Pmc and Pbf and can therefore be a better option for Arg side
chain protection. In addition, MIS is compatible with tryptophan-containing peptides.
Introduction
Most peptides synthesized on a solid-phase are prepared using
the Fmoc/tert-butyl strategy.1,2 Thus, a-amino temporary protec-
tion is achieved with the base labile 9-fluorenylmethoxycarbonyl
(Fmoc) group; amino acid side chains are protected by tri-
fluoroacetic acid (TFA)-labile protecting groups, usually tBu
derivatives; and the C-terminal amino acid is anchored to the
solid support through a TFA-labile linker/handle. Nevertheless,
tert-butyl-type protection of a number of amino acids is not the
best option because of factors such as inefficiency in preventing
side reactions or inadequate TFA lability. Among these amino
acids, protection of the basic guanidinium group of Arginine (Arg)
is possibly the most critical case.3
Currently, the most frequently used TFA-labile Arg-
protecting groups are based on electron-rich benzene sulfonyl
moieties.4 These groups are, in increasing order of acid labil-
ity: 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds),5 4-methoxy-
2,3,6-trimethylsulfonyl (Mtr),5 2,2,5,7,8-pentamethylchroman-6-
sulfonyl (Pmc),6,7 and 2,2,4,6,7-pentamethyldihydrobenzofurane-
5-sulfonyl (Pbf) (Fig. 1).8,9
Fig. 1 Protecting groups for the side-chain of Arg.
tection of Arg remains unsolved because even the Pbf group is too
stable to TFA and its removal requires high TFA concentrations
and long treatment times, which may not be appropriate for
acid-sensitive peptides. The conditions become increasingly more
demanding when preparing multiple Arg-containing peptides,
which show biological properties of great interest.11
All of these mask the reactivity of the Nw , are commercially
available and have been extensively used in the Fmoc/tBu solid-
phase strategy.10 Nevertheless, the problem of the side chain pro-
The design of a new sulfonyl-based Arg-protecting group is
not a straightforward process in the sense of simply adding
electron-donating groups to an aromatic ring, because the pla-
narity of the system, which is essential for TFA lability, is not
easily conserved because of the presence of the sulfonyl group.
Thus, trimethoxybenzenesulfonyl (Mtb), which contains more
electron-rich substituents (3 MeO) is less acid-labile than Mds
(1 MeO, 2 Me) and Mtr (1 MeO, 3 Me).6 This characteristic
is attributed to the loss of planarity caused by the presence of
the two methoxy groups near the sulfonyl group. Furthermore,
the sulfonyl derivative of 3,4-ethylenedioxythiophene (EDOT),
whose derived compounds are highly labile to TFA as carboxylic
acid protectors,12 is not labile as an Arg side-chain protector,
possibly because of the same loss of planarity.13 Common side-
reactions associated with the use of these benzenesulfonyl-based
protecting groups are arylation of sensitive residues, such as Trp,14
aInstitute for Research in Biomedicine, Barcelona Science Park, Baldiri
Reixac 10, 08028 Barcelona, Spain. E-mail: albericio@irbbarcelona.
org, mercedes.alvarez@irbbarcelona.org; Fax: +3493 4037126; Tel: +3493
4037088
bLonza AG., TIDES, CH-3930, Visp, Switzerland
cCIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona,
Spain
dLaboratory of Organic Chemistry, Faculty of Pharmacy, University of
Barcelona, 08028 Barcelona, Spain
eDepartment of Organic Chemistry, University of Barcelona, Mart´ı i
Franque´s 1, 08028 Barcelona, Spain
† Electronic supplementary information (ESI) available: Detailed exper-
imental procedures for intermediates, final Fmoc-Arg(MIS)-OH, and
peptides; 1H- and 13C-NMR spectra and HPLC chromatograms. See DOI:
10.1039/b904836g
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or sulfonation of Trp and/or Arg residues themselves.15 These
side reactions are favored by the decomposition of the sulfonyl-
protecting group in two moieties, the arylcarbocation, which is an
alkylating agent, and the sulfonyl part, which can cause addition
of sulfur trioxide on the peptidic chain.15
In an attempt to overcome the above mentioned drawbacks, we
describe herein a new more acid-labile Arg side chain-protecting
group based on the indole system.
Results and discussion
General
A TFA-labile protecting group should be based on an electron-rich
system. In this regard, N-alkylindole derivatives have been used
as acid-labile amide linkers16 and amide backbone protectors.17
Taking this into account, we chose 1,2-dimethylindole-3-sulfonyl
(MIS) as a guanidinium-protecting group (Fig. 1). The extra
methyl at position 2 should increase the acid lability of the
protecting group and prevent electrophilic aromatic substitution.
Furthermore, the 1,2-dimethylindole is commercially available.
As the 1,2-dimethylindole is prone to polymerization in strong
acidic conditions, sulfonation of the indole ring must be carried out
in neutral or basic media. Thus, chlorosulfonic acid, which is the
reagent of choice for Pmc and Pbf sulfonylation, cannot be used
in the case of 1,2-dimethylindole. Nevertheless, the use of sulfur
trioxide pyridine complex yielded the corresponding pyridinium
sulfonate in good yield.18 Chlorination under mild conditions
by treatment with oxalyl chloride yielded 1,2-dimethylindole-3-
sulfonyl chloride (MIS-Cl). These conditions gave much better
overall yield (80%) to those attained with Pbf and Pmc (51% and
53% respectively),6,8 with the advantage that 1,2-dimethylindole is
commercially available (Scheme 1).
Scheme 1 Synthetic pathway for the preparation of Fmoc-Arg(MIS)-OH.
These assays revealed that the MIS group is considerably more
acid-labile than the Pbf one.
Also, the MIS derivative generated in the removal process
differs from the case of Pbf. For Pbf and Pmc, 2,2,5,7,8-
pentamethylchroman and 2,2,4,6,7-pentamethyldihydrobenzo-
furane, respectively, are formed via a desulfonylation mechanism,6
while for MIS, the sulfonic acid (MIS-OH) was stable and was not
desulfonated.
Synthesis of multiple arginine-containing peptides using MIS and
Pbf protection
We prepared Fmoc-Arg(MIS)-OH in a similar way to Pmc/Pbf
derivatives,6,8 using Z-Arg-OH as starting material. Z-Arg-OH was
sulfonylated at the Nw position with MIS-Cl and the Z group
was removed via catalytic hydrogenolysis. Final Fmoc protection
was achieved by using Fmoc-2-mercaptobenzothiazole (Fmoc-2-
MBT) because the use of other more active Fmoc derivatives leads
to the formation of dipeptides or other side reactions.19,20
As Pbf removal is more complicated in multiple Arg-containing
peptides, Ac-Phe-Arg-Arg-Arg-Arg-Val-NH2 was chosen as a
model peptide to compare the acid lability of MIS and Pbf.8,21 The
corresponding Pbf- and MIS-protected peptides were prepared
using standard solid-phase peptide synthesis protocols on Sieber
amide resin, which allows cleavage from the resin with small
amounts of TFA (2%), thereby yielding the MIS- and Pbf-
protected peptides respectively with excellent purity.
Optimization of the scavengers used in the removal
As MIS-OH is a polar compound, it precipitates during the
ether treatment after the cleavage step. Alternative scavengers
to H2O were tested to reduce the amounts of the strongly UV
absorbant MIS-OH in order to facilitate purification. Among the
scavengers tested, the optimum were 10% 3,4-dimethoxyphenol,
1,3,5-trimethoxybenzene (Tmb) or 3,5-dimethoxyphenol. The use
of these scavengers reduced the amounts of MIS-OH more than
10 fold (40 times in the case of Tmb), thereby simplifying HPLC
purification to yield the final product.
Synthesis of Trp-containing peptides
To check the compatibility of the MIS group with Trp,22 we
first synthesized the model peptides Z-Arg(MIS)-Trp(Boc)-Ala-
Gly-NH2 and Z-Arg(Pbf)-Trp(Boc)-Ala-Gly-NH2 on a Sieber
amide resin, which were obtained with an excellent HPLC
purity. Afterwards, both resins were treated with TFA-CH2Cl2-
trimethoxybenzene (50 : 40 : 10) to compare the purities of
Removal assays
To compare the acid lability of the Pbf group, which is more acid-
labile than Pmc, with the MIS group, protected peptide-bonded
resins were treated with TFA–CH2Cl2–H2O–TIS (50 : 45 : 2.5 : 2.5)
for 30 (MIS, 100%; Pbf, 4%) and 60 min (MIS, 100%; Pbf, 38%).
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Trp-containing peptides after MIS and Pbf removal. Trp alkyla-
tion or sulfonation was not detected in either case. The purity
of the crude product was greater in the case of MIS and
neither the MIS-protected peptides nor MIS-OH were detected
by LC-MS. Nevertheless, in the case of the Pbf experiment,
considerable amounts of the Pbf-protected peptide were detected
(34% compared to unprotected peptide, HPLC, l = 220 nm).
In summary, MIS is the most acid-labile sulfonyl-type protecting
group for Arg described to date. This feature makes it highly
convenient for the synthesis of multiple Arg-containing peptides
or peptides that contain acid-sensitive moieties. Furthermore, MIS
is compatible with Trp-containing peptides.
HRMS (CI): m/z calcd. for C10H10NO2S [M–Cl]- 208.0426, found
208.0427.
Z-L-Arg(MIS)-OH (3). Z-L-Arg-OH (2.05 g, 6.7 mmol) was
suspended in 3 N aqueous NaOH (6.7 mL, 20 mmol) and acetone
(13.3 mL) was added to dissolve the product. The reaction was
cooled in an ice bath and 3 N aqueous NaOH (6.7 mL) and a
solution of compound 2 (3.69 g, 14.7 mmol) in acetone (13.3 mL)
were simultaneously added over 10 min. The reaction mixture was
stirred at 0 ◦C for 2 h and at room temperature for a further
2 h. After that time, starting material 2 was no longer detected
by TLC (hexane–EtOAc, 1 : 1). H2O (100 mL) was added and
the suspension was washed with diethyl ether (3 ¥ 80 mL). The
aqueous phase was acidified to pH 2–3 by addition of 1 N HCl, the
precipitate obtained was filtered, washed with acidic water (pH 2–
3) and dried in vacuo. The crude product obtained was purified
by column chromatography (CH2Cl2, MeOH, 1% HOAc). The
solvent of the pure fractions was removed in vacuo to yield an
oil. This process was repeated. Hexane and CH2Cl2 were then
sequentially added and a precipitate appeared on scratching. The
solvent was decanted and the solid was washed 4 times with
CH2Cl2–hexane (enough hexane to precipitate all the product)
to remove HOAc and give 3 (0.70 g, 20.4% yield).24 Mp = 155.5–
159.1 ◦C.1H NMR (400 MHz, DMSO-d-6): d = 7.85 (d, 1H, CH,
J = 7.6 Hz), 7.52 (d, 1H, NH, J = 8.0 Hz), 7.43 (d, 1H, CH, J =
8.0 Hz), 7.30 (m, 5H, 5CH Z), 7.10 (m, 2H, 2CH), 5.01 (s, 2H,
CH2), 3.87 (m, 1H, aCH), 3.66 (s, 3H, CH3), 3.0 (m, 2H, CH2),
2.60 (s, 3H, CH3), 1.64 (m, 1H, CH2), 1.49 (m, 1H, CH2), 1.41
(m, 2H, CH2).13C NMR (100 MHz, DMSO-d-6): d = 174.4 (C),
157.0 (C), 156.8 (C), 139.4 (C), 137.7 (C), 135.9 (C), 129.0 (CH),
128.5 (CH), 128.4 (CH), 125.2 (C), 122.1 (CH), 121.1 (CH), 120.1
(CH), 110.4 (CH), 66.1 (CH2), 54.3 (CH), 40.0 (CH2), 30.2 (CH3),
28.9 (CH2), 26.4 (CH2), 11.4 (CH3). HRMS (CI): m/z calcd. for
C24H30N5O6S [M + H]+ 516.1911, found 516.1911.
Experimental section
Synthesis of the protecting group and Arginine protection
Pyridinium 1,2-dimethylindole-3-sulfonate (1). 1,2-Dimethyl-
indole (14.5 g, 99.8 mmol) and sulfur trioxide pyridine complex
(19.1 g, 119.8 mmol) were dissolved in pyridine (70 mL) under
an Ar atmosphere. The reaction mixture was refluxed for 1 h
and HPLC indicated the reaction was complete (99.2% HPLC
◦
conversion, 254 nm). The reaction mixture was cooled to 60 C
and concentrated under vacuum to give a solid. The crude product
was used directly for the next step.23 1H NMR (400 MHz, D2O):
d = 8.44 (d, 2H, 2CH, J = 5.8 Hz), 8.31 (m, 1H, CH), 7.75
(m, 2H, 2CH), 7.67 (d, 1H, CH, J = 7.7 Hz), 7.14 (d, 1H, CH,
J = 7.4 Hz), 7.05 (m, 2H, 2CH), 3.38 (s, 3H, CH3), 2.41 (s, 3H,
CH3).13C NMR (100 MHz, D2O): d = 147.0 (CH), 140.9 (CH),
139.2 (C), 135.6 (C), 127.3 (CH), 124.1 (C), 122.0 (CH), 121.0
(CH), 119.2 (CH), 112.8 (C), 109.9 (CH), 29.2 (CH3), 10.4 (CH3).
HRMS (CI): m/z calcd. for C10H10NO3S [M–H]+ 224.0386, found
224.0388.
1,2-Dimethylindole-3-sulfonyl chloride (MIS-Cl) (2). All the
crude product 1 obtained in the previous step was suspended
in dry CH2Cl2 (200 mL) under Ar atmosphere. The suspension
was cooled in an ice bath and oxalyl chloride (20.0 g, 158 mmol)
was slowly added. DMF (0.5 mL) was then slowly and carefully
added and vigorous effervescence was observed. The reaction
mixture was stirred in an ice bath for a further 30 min until the
effervescence ceased and was then stirred at room temperature
for 2 h. An aliquot (6 mL) was then treated with MeOH for
20 min and injected into the HPLC apparatus, which showed the
presence of methyl 1,2-dimethylindole-3-sulfonate and an absence
of starting material. CH2Cl2 (200 mL) was added to the reaction
mixture and it was cooled to below 5 ◦C. H2O (2–8 ◦C, 150 mL)
was added and the mixture was stirred for 10 min. The organic
phase was separated and washed with 2–8 ◦C H2O (2 ¥ 150 mL),
and dried with anhydrous MgSO4 (15 g). The solution was then
concentrated to ca. 40 mL. The solid was filtered off and washed
with CH2Cl2–n-hexane (1 : 1, 60 mL). The solid was dried under
vacuum to give light pink solid (19.6 g, 80.4% yield◦, 98% HPLC
purity, base on 1,2-dimethylindole). Mp = 67.7–73.5 C. 1H NMR
(400 MHz, DMSO-d-6): d = 7.82 (d, 1H, CH, J = 7.8 Hz),
7.36 (d, 1H, NH, J = 8.0 Hz), 7.08 (m, 2H, 2CH), 7.00 (m, 2H,
2CH), 3.63 (s, 3H, CH3), 2.56 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d-6): d = 137.2 (C), 135.9 (C), 125.5 (C), 121.4 (CH),
120.8 (CH), 120.1 (CH), 109.7 (CH), 30.0 (CH3), 11.3 (CH3).
H-L-Arg(MIS)-OH (4). A mixture of 3 (486 mg, 0.94 mmol)
and Pd/C (10%) (110 mg) in MeOH (60 mL) was hydrogenated
overnight at atmospheric pressure. After this time, TLC (CH2Cl2–
MeOH–HOAc, 90:9:1) still showed some starting material. More
10% Pd/C (100 mg) was added and the mixture was hydrogenated
for a further 24 h, after which TLC showed the absence of
starting material. The reaction mixture was filtered over celite
and evaporated to dryness to yield 4 (352 mg, 98% yield). Mp =
◦
153.2–155.0 C.1H NMR (400 MHz, DMSO-d-6): d = 7.83 (d,
1H, CH, J = 7.6 Hz), 7.47 (d, 1H, NH, J = 8.1 Hz), 7.42 (d, 1H,
CH, J = 8.1 Hz), 7.11 (m, 2H, 2CH), 3.65 (s, 3H, CH3), 3.17 (m,
1H, CH), 3.00 (m, 2H, CH2), 2.60 (s, 3H, CH3), 1.65 (m, 1H, CH2),
1.54 (m, 1H, CH2), 1.42 (m, 2H, CH2). HRMS (CI): m/z calcd.
for C16H24N5O4S [M + H]+ 382.1544, found 382.1542.
Fmoc-Arg(MIS)-OH (5). H-Arg(MIS)-OH (250 mg,
0.658 mmol) was suspended in 1% aqueous Na2CO3 (2 mL).
1,4-dioxane (2 mL) was added and the product was dissolved. The
pH was basified to 9–10 with saturated aqueous Na2CO3 (300 mL
in our case). Fmoc-2-mercaptobenzotiazole (Fmoc-2-MBT)
(256 mg, 0.658 mmol) in 1,4-dioxane (700 mL) was slowly added.
The pH was kept between 9 and 10 with saturated aqueous
Na2CO3 and the resulting suspension was stirred overnight. After
14 h of reaction, H2O (9 mL) was added, the pH was neutralized
with 1 N HCl and the solution was washed with tert-butylmethyl
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ether (3 ¥ 5 mL). The aqueous phase was acidified to pH 2–3
with 1 N HCl and extracted with EtOAc (3 ¥ 7 mL). Note that
to dissolve the precipitated product vigorous stirring is required.
The organic phases were pooled, dried over dry MgSO4, filtered
and evaporated to dryness, thereby yielding a solid. Various
co-evaporations with CH2Cl2 were performed to yield the desired
product as a solid (207 mg, 52% yield). Mp = 137.2–146.4 ◦C. 1H
NMR (400 MHz, DMSO-d-6): d = 7.86 (m, 3H, 3CH), 7.70 (d,
2H, 2CH, J = 7.4 Hz), 7.59 (d, 1H, NH, J = 7.9 Hz), 7.42 (d, 1H,
CH, J = 8.1 Hz), 7.39 (m, 2H, 2CH), 7.30 (m, 2H, 2CH), 7.10 (m,
2H, 2CH), 4.27 (m, 2H, CH2), 4.20 (m, 1H, CH), 3.86 (m, 1H,
CH), 3.66 (s, 3H, CH3), 3.01 (m, 2H, CH2), 2.61 (s, 3H, CH3), 1.65
(m, 1H, CH2), 1.52 (m, 1H, CH2), 1.38 (m, 2H, CH2). 13C NMR
(100 MHz, DMSO-d-6): d = 174.4 (C), 157.0 (C), 156.8 (C), 144.5
(C), 141.4 (C), 139.4 (C), 135.9 (C), 128.3 (CH), 127.8 (CH),
126.0 (CH), 125.2 (C), 122.1 (CH), 121.1 (CH), 120.8 (CH), 120.1
(CH), 110.4 (CH), 66.3 (CH2), 55.6 (CH), 47.3 (CH), 40.0 (CH2),
30.2 (CH3), 28.8 (CH2), 26.5 (CH2), 11.4 (CH3). HRMS (CI): m/z
calcd. for C31H34N5O6S [M + H]+ 604.2224, found 604.2222.
LC-MS and HRMS (CI): m/z calcd. for C92H136N19O19S4 [M +
H]+ 1938.9137, found 1938.9202.
Removal assays. General procedure: the resin (3 mg) was treated
with cleavage solution (50 mL). After the cleavage time, the
solution was poured into H2O (4 mL), and TFA and CH2Cl2
were evaporated. The resulting aqueous solution was washed with
CH2Cl2 (6 ¥ 1 mL), frozen, lyophilized and analyzed by HPLC
(l = 220 nm) and ESMS or MALDI-TOF.
Optimization of the scavengers. The same procedure as for the
removal assays was followed. In all the experiments the resin
was treated with TFA-CH2Cl2-scavenger (50 : 40 : 10) (50 mL)
for 1 h. The scavengers tested were 3,4-dimethoxyphenol, 1,3,5-
trimethoxybenzene (Tmb) or 3,5-dimethoxyphenol.
Z-Arg-Trp-Ala-Gly-NH2 (model peptide 2).
Z-Arg(MIS)-Trp(Boc)-Ala-Gly-NH2
and
Z-Arg(Pbf)-
Trp(Boc)-Ala-Gly-NH2. Sieber amide resin (70 mg, 0.40 mmol/g)
was placed in a 2 mL polypropylene syringe fitted with a
polyethylene filter disc. The resin was swollen with CH2Cl2,
washings with CH2Cl2 and DMF were carried out and the Fmoc
group was removed. Fmoc-L-Gly-OH (33.3 mg, 112 mmol),
Fmoc-L-Ala-OH (34.9 mg, 112 mmol) and Fmoc-L-Trp(Boc)-OH
(59.0 mg, 112 mmol) were sequentially coupled using PyBOP
(58.3 mg, 112 mmol) HOAt (15.2 mg, 112 mmol) and DIPEA
(57.4 mL, 336 mmol) in DMF, t = 1.5 h. The resin was divided
into two equal parts.
Synthesis of arginine-containing model peptides using MIS and
Pbf protection
Ac-Phe-Arg-Arg-Arg-Arg-Val-NH2 (model peptide 1).
Ac-Phe-Arg(MIS)-Arg(MIS)-Arg(MIS)-Arg(MIS)-Val-NH2.
Sieber amide resin (25 mg, 0.42 mmol/g) was placed in a 2 mL
polypropylene syringe fitted with a polyethylene filter disc. The
resin was swollen with CH2Cl2, washings with CH2Cl2 and
DMF were carried out and the Fmoc group was removed by
treatment with piperidine–DMF (2 : 8) (1 ¥ 1 min, 2 ¥ 10 min).
Fmoc-L-Val-OH (14.3 mg, 42.1 mmol) was coupled using HOBt
(5.7 mg, 42.1 mmol) and DIC (6.7 mL, 42,1 mmol) in DMF, t =
90 min. The Fmoc group was removed in the usual way and Fmoc-
L-Arg(MIS)-OH (15.8 mg, 26.3 mmol) was coupled using PyBOP
(13.7 mg, 26.3 mmol) HOAt (3.6 mg, 26.3 mmol) and DIPEA
(13.4 mL, 78.9 mmol) in DMF for 90 min. The resin was acetylated
by treatment with Ac2O (50 eq.) and DIPEA (50 eq.) in DMF for
25 min. The Fmoc group was removed and the same procedure
was repeated three more times, acetylating the resin before
each Fmoc removal. After the last Fmoc removal, Fmoc-L-Phe
(13.6 mg, 35 mmol) was coupled using PyBOP (18.3 mg, 35 mmol)
HOAt (4.8 mg, 35 mmol) and DIPEA (17.9 mL, 105.2 mmol) in
DMF for 90 min. The Fmoc group was removed and the resulting
free amino group was acetylated as before. The resin was washed
with DMF, CH2Cl2 and diethyl ether, dried in vacuo, and divided
into five aliquots. One of these was swollen with CH2Cl2, and
treated with 1.5 mL of TFA–CH2Cl2–TIS–H2O (2 : 93 : 2.5 : 2.5)
for 20 min in order to cleave the protected peptide from the resin.
The resin was filtered and the solution collected was diluted with
CH2Cl2 and neutralised by adding DIPEA (80 mL, 1.2 eq. per eq.
of TFA). The solvent was then removed in vacuo, and H2O and
AcCN were added and the solution was frozen and lyophilized.
The product obtained was characterised by LC-MS and HRMS
(CI): m/z calcd. for C80H107N23O15S4 [M + Na]+ 1780.7092, found
1780.7152.
Part 1 [Z-Arg(MIS)-Trp(Boc)-Ala-Gly-NH2]. Z-Arg(MIS)-
OH (28.9 mg, 56 mmol) was coupled using PyBOP (29.2 mg,
56 mmol) HOAt (7.6 mg, 56 mmol) and DIPEA (28.7 mL, 168 mmol)
in DMF, t = 1.5 h. The resin was washed with DMF, CH2Cl2 and
diethyl ether, dried in vacuo and divided into 4 mg aliquots. One of
them was swollen with CH2Cl2 and treated with 1.5 mL of TFA–
CH2Cl2–TIS–H2O (2 : 93 : 2.5 : 2.5) for 20 min in order to cleave
the protected peptide from the resin. The resin was filtered and
the collected solution was diluted with CH2Cl2 and neutralised by
adding DIPEA (80 mL, 1.2 eq. per eq. of TFA). The solvent was
then removed in vacuo, and H2O and AcCN were added and the
solution was frozen and lyophilized. The product obtained was
characterised by LC-MS (95% purity). HRMS (CI): m/z calcd.
for C45H57N10O10S [M + H]+ 929.3974, found 929.3969.
Part
2
[Z-Arg(Pbf)-Trp(Boc)-Ala-Gly-NH2]. Fmoc-
Arg(Pbf)-OH (36.3 mg, 56 mmol) was coupled using PyBOP
(29.2 mg, 56 mmol), HOAt (7.6 mg, 56 mmol) and DIPEA
(28.7 mL, 168 mmol) in DMF, t = 1.5 h. The Fmoc group was
removed and the free amine was protected with the Z group by
treatment with Z-OSu (14.0 mg, 56 mmol) and DIPEA (35.9 mL,
210 mmol). The resin was then washed with DMF, CH2Cl2 and
diethyl ether, dried in vacuo, and divided into 4 mg aliquots, one
of which was cleaved in the same way as for Part 1. The product
obtained was characterised by LC-MS (96% purity).
Z-Arg-Trp-Ala-Gly-NH2 from Z-Arg(MIS)-Trp(Boc)-Ala-
Gly-NH2. Two aliquots from Part 1 were treated with TFA–
CH2Cl2–1,3,5-trimehtoxybenzene (50 : 40 : 10) and TFA–CH2Cl2–
H2O (50 : 45 : 5) respectively for 1 h following the General
procedure for the removal assays described above. In the latter
case, no CH2Cl2 washings were performed. The two crude products
resulting from these treatments were analyzed by LC-MS. No
Ac-Phe-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Val-NH2. The
same procedure as for the synthesis of peptide 1 was used but
replacing Fmoc-L-Arg(MIS)-OH by Fmoc-L-Arg(Pbf)-OH
(17.1 mg, 26.3 mmol). The product obtained was characterised by
2568 | Org. Biomol. Chem., 2009, 7, 2565–2569
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Trp alkylation or sulfonation nor MIS-protected peptide were
observed.
Soc. C., 1971, 46w,w¢-bis-Boc (ref. 4) as well as the 10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5-yl [5-dibenzosuberyl] (Sub) (M. Noda and
M. Kiffe, J. Pept. Res., 1997, 50, 329), have been also proposed but not
broadly used.
Z-Arg-Trp-Ala-Gly-NH2 from Z-Arg(Pbf)-Trp(Boc)-Ala-Gly-
NH2. An aliquot from Part 2 was treated with TFA-CH2Cl2-
trimehtoxybenzene (50 : 40 : 10) for 1 h following the General
procedure for the removal assays described above. The target pep-
tide was analyzed by LC-MS (60% purity). 17% of Pbf- protected
peptide was detected and no Trp alkylation or sulfonation was
observed.
11 S. Futaki, I. Nakase, A. Tadokoro, T. Takeuchi and A. T. Jones,
Biochem. Soc. Trans., 2007, 35, 784.
´
12 A. Isidro-Llobet, M. Alvarez and F. Albericio, Tetrahedron Lett., 2008,
49, 3304.
´
13 A. Isidro-Llobet, M. Alvarez, and F. Albericio, unpublished results.
14 P. Sieber, Tetrahedron Lett., 1987, 28, 1637.
15 A. Beck-Sickinger, G. Schnorrenberg, J. Metzger and G. Jung,
Int. J. Pept. Prot. Res., 1991, 38, 25.
16 K. G. Estep, C. E. Neipp, L. M. Stramiello, A. Stephens, D. Mavis,
M. P. Allen, S. Robinson and E. J. Roskamp, J. Org. Chem., 1998, 63,
5300.
Acknowledgements
´
17 A. Isidro-Llobet, X. Just-Baringo, M. Alvarez and F. Albericio,
Biopolymers, 2008, 90, 444.
This work was partially supported by CICYT (CTQ2006–
03794/BQU), the Instituto de Salud Carlos III (CB06_01_0074),
the Generalitat de Catalunya (2005SGR 00662), the Institute for
Research in Biomedicine, and the Barcelona Science Park. AI-L
thanks the DURSI, Generalitat de Catalunya, and the European
Social Funds for a predoctoral fellowship.
18 G. F. Smith and D. A. Taylor, Tetrahedron, 1973, 29, 669.
´
19 A. Isidro-Llobet, X. Just-Baringo, A. Ewenson, M. Alvarez and F.
Albericio, Biopolymers, 2007, 88, 733.
20 For a broad discussion of this subject, see: L. J. Cruz, N. G. Beteta, A.
Ewenson and F. Albericio, Org. Process Res. Dev., 2004, 8, 920.
21 B. Riniker, A. Hartman, Pept.: Chem., Struct. Biol., Proc. Am. Pept.
Symp., 11th, ed. J. Rivier, and G. R. Marshall, Escom, Sci. Pub. Leiden,
The Netherlands, 1990, pp. 950–952.
22 A. Stierandova, N. F. Sepetov, G. V. Nikiforovich and M. Lebl,
Int. J. Pept. Prot. Res., 1994, 43, 31.
Notes and references
23 In other experiments the final product was isolated using the following
work up: The reaction mixture was cooled to room temperature and
H2O was added (400 mL). The resulting solution was washed with
diethyl ether (4 ¥ 250 mL). The aqueous phase was evaporated to
dryness and dried in the vacuum dessicator to render a red oily solid
(96% yield), which was characterized by NMR and HRMS.
1 P. Lloyd-Williams, F. Albericio, and E. Giralt, Chemical Approaches to
the Synthesis of Peptides and Proteins, CRC Press, Boca Raton, New
York, 1997.
2 T. Cupido, J. Tulla-Puche, J. Spengler and F. Albericio, Curr. Opin.
Drug Dis. Develop., 2007, 10, 768.
24 A similar procedure which avoids the column chromatography purifi-
cation and may be more convenient for industrial scale has also been
developed: Z-L-Arg-OH (5.0 g, 16.2 mmol) was dissolved in acetone
(160 mL) and 3 N aqueous NaOH (45 mL, 135 mmol). The reaction
was cooled in an ice bath and compound 2 (3.85 g, 15.8 mmol) dissolved
in acetone (100 mL) was added over 10 min. The reaction mixture was
stirred for 1 h at 0 ◦C. Additional, 2 (1.9 g, 7.8 mmol) in acetone (50 mL)
was then added followed by 90 min of stirring at 0 ◦C. Finally, a last
portion of compound 2 (1.9 g, 7.8 mmol) in acetone (50 mL) was added
and the reaction mixture was stirred at 0 ◦C for additional 30 min and
at room temperature for 3 h. At this point, species 2 was no longer
observed by HPLC. Acetone, was distilled out under vacuum and H2O
(100 mL) was added (pH = 14). Then EtOAc (150 mL) was added
and the pH adjusted to 3.0 with 50% aqueous citric acid. The solution
became 3 phases. The above organic phase as well as the underneath oil
phase were separated. H2O (30 mL) was added to the oil phase, and the
pH adjusted to 10. To make the solution more homogeneous, EtOAc
(50 mL) was added and the pH adjusted to 3.0. The EtOAc phase was
separated and combined with the EtOAc phase from the first extraction.
The combining solution was concentrated to 100 mL. H2O (100 mL)
was added, the pH was adjusted firstly to 10 and finally to 3.0. At this
point, the aqueous phase was discharged. This process was repeated
and the EtOAc solution was concentrated to give a light yellow solid
(2.9 g, yield: 24.5%, HPLC purity: 70.6%).
3 P. Thamm, W. Kolbeck, H.-J. Musiol, and L. Moroder, Synthesis
of Peptides and Peptidomimetics, Houben-Weyl Methods in Organic
Chemistry, ed. M. Goodman, A. Felix, L. Moroder, and C. Toniolo,
vol E 22a, pp. 315-333.
4 w,w¢-bis-Boc Arginine derivatives have a similar acid lability to TFA as
Pbf but are not so widely used. The coupling rates of bis-Boc protected
Arg are low. B. F. Lundt, N. L. Johansen, A. Volund and J. Markussen,
Int. J. Pept. Protein Res., 1978, 12, 258; A. S. Verdini, P. Lucietto, G.
Fossati and C. Giordani, Tetrahedron Lett., 1992, 33, 6541.
5 M. Fujino, M. Wakimasu and C. Kitada, Chem. Pharm. Bull., 1981,
29, 2825.
6 R. Ramage, J. Green and A. J. Blake, Tetrahedron, 1991, 47,
6353.
7 I. M. Eggleston, J. H. Jones and P. Ward, J. Chemical Research (S),
1991, 10, 286.
8 L. A. Carpino, H. Shroff, S. A. Triolo, E. M. E. Mansour, H. Wenschuh
and F. Albericio, Tetrahedron Lett., 1993, 34, 7829.
9 C. G. Fields and G. B. Fields, Tetrahedron Lett., 1993, 42, 6661.
10 The use of the w-NO2 protected arginine derivative (M. Bergmann, L.
Zervas and H. Rinke, H-S Z. Physiol. Chem, 1934, 224, 40) which is also
commercially available, has the main drawback of long reaction times,
which is an inconvenient in the case of sensitive peptides. For instance in
the case of hydrogenolysis partial hydrogenation of Trp or even Phe can
occur; G. T. Young, D. J. Schafer, D. F. Elliott and R. Wade, J. Chem.
This journal is
The Royal Society of Chemistry 2009
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