Synthesis of an arginine tagged [Cys155-Arg180] fragment of NY-ESO-1: elimination of an undesired by-product using 'In house' resins

Article abstract of DOI:10.1055/s-0029-1216952

During the solid-phase synthesis of a peptide fragment derived from the cancer protein NY-ESO-1 incorporating a solubilising arginine tag, a significant by-product was obtained that lacked the arginine tag. The formation of this by-product (and others) wa

Full text of DOI:10.1055/s-0029-1216952

3460
PAPER
Synthesis of an Arginine Tagged [Cys155–Arg180] Fragment of NY-ESO-1:
Elimination of an Undesired By-product Using ‘In House’ Resins
S
a
a[Cys –A 180
r
55 u
]
Fragm
e
l
nt of NY-ESO
W
-1 . R. Harris,* Margaret A. Brimble*
Department of Chemistry, University of Auckland, 23 Symonds St, Auckland 1142, New Zealand
Fax +64(9)3737422; E-mail: paul.harris@auckland.ac.nz; E-mail: m.brimble@auckland.ac.nz
Received 5 June 2009
[154–180] was required as one component of a subse-
Abstract: During the solid-phase synthesis of a peptide fragment
derived from the cancer protein NY-ESO-1 incorporating a solu-
bilising arginine tag, a significant by-product was obtained that
lacked the arginine tag. The formation of this by-product (and oth-
quent native chemical ligation reaction10,11 used to prepare
the final protein molecule. The hydrophobic nature of the
sequence necessitated incorporation of an arginine tag in
ers) was highly dependant on the quality of the resin used and was order to facilitate its handling and solubility.
completely removed when the resin was synthesized ‘in house’.
We herein compare our results for the preparation of a
peptide containing a C-terminal arginine tag prepared by
Key words: solid-phase, resins, HMBA linker, arginine tag
Fmoc SPPS using either commercially available Wang,
Rink or 2-chlorotrityl chloride resin with aminomethyl
resin functionalized with an appropriate linker that was
prepared in house.
9-Fluorenylmethoxycarbonyl (Fmoc) based solid-phase
peptide synthesis (SPPS) has arguably become the meth-
od of choice for the routine synthesis of polypeptides.1
While variables such as deprotection, coupling and cleav-
age procedures used during Fmoc SPPS are undoubtedly
important factors, probably the most crucial factor for
successful solid-phase synthesis is the quality of the solid
support.2,3
HO
linker
PS-1% DVB
Fmoc-Xaa-CO2H path A
O
Xaa-Fmoc
C
O
linker
The preparation of a solid support or resin containing a
suitable linker and/or the first Fmoc amino acid can gen-
erally be divided into two chemistries (Scheme 1). Firstly
(path A), the base resin, which is typically formed from
co-polymerisation of polystyrene (PS) with 1% divinyl-
benzene, can be subsequently modified in a series of
chemical steps that take place entirely on the resin, lead-
ing to resin functionalized with linkers such as Wang,4 2-
chlorotrityl chloride5 or Rink Amide.6 In this case little
analytical control can be carried out to ensure chemical in-
tegrity at each step. Thereafter, the C-terminal Fmoc ami-
no acid is introduced, which can lead to undesired side
reactions such as racemization,7 dipeptide formation,8 or
low levels of substitution. Alternatively, the resin contain-
ing a suitable functional group such as an aminomethyl
(H2NCH2-) can be reacted with an Fmoc amino acid that
is already attached to the requisite linker (path B). The lat-
ter method is the method of choice for SPPS since both the
aminomethyl resin and the Fmoc-amino acid with the ap-
propriate linker, can be easily synthesized, purified and
characterized prior to condensation.9 In addition, the cou-
pling of the Fmoc amino acid linker conjugate to the ami-
nomethyl resin can be accomplished using normal
methods for peptide bond formation.
H2N
O
C
path B
linker
OH
Fmoc-Xaa
O
linker
C N
Fmoc-Xaa
H
Scheme 1 Two strategies for the synthesis of Fmoc-based SPPS
resins; Xaa = any amino acid
It was decided to use a tag consisting of six arginine resi-
dues joined to the C-terminus by a 4-hydroxymethylben-
zioc acid (HMBA) linker,12 which is stable to the
conditions used for Fmoc SPPS (including acidic cleav-
age) but can be cleaved under basic conditions with vari-
ous nucleophiles.13 Using commercially available Fmoc-
Arg(Pbf)-Wang PS resin 1 and microwave (MW) Fmoc
SPPS protocols, Fmoc-Arg(Pbf)6-Wang PS resin 2 was
prepared using 20% piperidine for Fmoc deprotection and
HBTU/DIPEA as coupling reagents (Scheme 2). Cleav-
age of a small sample confirmed the expected product as
Fmoc-Arg6-OH (19; see experimental section). Deprotec-
tion of the resin-bound N-terminal Fmoc, followed by
DIC/HOBt-mediated coupling of HMBA manually for
two hours gave a negative ninhydrin test. LC-MS analysis
of a cleaved resin sample established that the expected
product HOCH2-C6H4-CONH-[Arg]6-OH was the major
product (see experimental section).
The C-terminus of the cancer protein NY-ESO-1, NH2-
CQLSLL-Nle-WITQC(t-Bu)FLPVFLAQPPSGQR-R-OH
SYNTHESIS 2009, No. 20, pp 3460–3466
x
x.
x
x
.
2
0
0
9
Advanced online publication: 21.08.2009
DOI: 10.1055/s-0029-1216952; Art ID: P08009SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of a [Cys155–Arg180] Fragment of NY-ESO-1
3461
Coupling of the first amino acid of the NY-ESO-1 se- ined, including reagent K,17 reagent R18 and reagent B,19
quence [Cys155-Cys(tBu)165-Arg180], namely Arg180, was as well as modifying the water content of the cleavage
undertaken manually with Fmoc-Arg(Pbf)-OH using cocktail. In all cases, the amount of cleavage at the linker
DIC/DMAP for one hour, and repeated once due to forma- was 18–20%. Extending the cleavage times from two
tion of the unreactive d-lactam during coupling.14 The res- hours to overnight did not increase the amount of by-prod-
in was then reacted with acetic anhydride/DMAP to cap uct formed, suggesting that the HMBA linker was inher-
any remaining hydroxymethyl functionality. In order to ently stable to the cleavage conditions.
assess peptide integrity and the extent of loading of the
O
first residue, a small amount of the resin 4 was cleaved
O
C
O
C
O
with TFA–TIS–H2O (95:2.5:2.5, v/v/v). In this instance,
the N-terminal Fmoc group was not removed in order to
facilitate retention of peptides on the reverse phase col-
umn. LC-MS revealed the expected product Fmoc-Arg-
HMBA-Arg6-OH (5), together with a later eluting by-
product (20%) that was determined to be Fmoc-Arg-OH15
(6) apparently resulting from hydrolysis at the HMBA
linker.16
NH-[Arg(Pbf)]6
O
Fmoc-Arg(Pbf)
4
Wang
1. MW Fmoc SPPS
2. TFA–TIS–EDT–H2O (95:1:2.5:2.5)
O
O
O
H2N
154–180
154–180
C-Arg6-CO2H
O
Fmoc-Arg(Pbf)
C
O
Wang
7, 80%
8, 20%
1
O
OH
1. 20% piperidine, DMF, MW
2. Fmoc-Arg(Pbf)-OH
HBTU, DIPEA, DMF, MW
H2N
repeat 5 x
Scheme 3 Fmoc SPPS preparation of NY-ESO-1 (155–180) cou-
pled to an arginine tag
O
Fmoc-Arg(Pbf)
C
O
Wang
2
We therefore explored the possibility that, during cou-
pling of the HMBA linker, some polymerisation takes
place on the resin; this was considered probable given that
DIC/HOBt was used for the initial coupling and these re-
agents can be used for ester bond formation. Such poly-
merisation may alter the reactivity at the linker. Hence,
following coupling of HMBA to the free N-terminal
amine of the H2N-Arg(Pbf)6-resin to give 3, hydrolysis,
methanolysis or hydrazinolysis was attempted with 0.1M
NaOH, MeOH/DIPEA or 5% hydrazine, respectively, in
order to remove any undesired polymerisation. Thereaf-
ter, Fmoc-Arg(Pbf)-OH was coupled as described in
Scheme 2 to give 4 and a small resin sample was cleaved
using TFA–TIS–H2O (95:2.5:2.5, v/v/v). In all three cas-
es, the amount of by-product Fmoc-Arg present in the
mixture ranged from 16–20%, indicating that no polymer-
isation took place during the coupling of the HMBA moi-
ety.
1. 20% piperdine, DMF
2. DIC, HOBt,
2 h
HO
CO2H
O
O
C
O
HO
NH-[Arg(Pbf)]6
C
3
Wang
1. Fmoc-Arg(Pbf)-OH
DIC, DMAP, 1 h
2. Ac2O, DMAP, 30 min
repeat 2 x
O
O
C
O
O
NH-[Arg(Pbf)]6
O
Fmoc-Arg(Pbf)
C
4
TFA–TIS–H2O (95:2.5:2.5)
2 h
O
O
O
O
OH
Fmoc-Arg
We also examined the coupling protocols used for the
condensation of HMBA to the N-terminus. Using HBTU/
DIPEA, or DIC/HOBt in either a large excess or stoichio-
metrically, did not significantly alter the amount of Fmoc-
Arg-OH released following acylation with Fmoc-
Arg(Pbf)-OH and subsequent TFA-mediated cleavage.
Fmoc-Arg
C-NH-Arg6-CO2H
+
5, 80%
6, 20%
Scheme 2 Preparation of an arginine tag coupled via an HMBA lin-
ker
In control experiments using non-microwave conditions
for the conversion of 1 into 2, or using a different batch of
the same pre-loaded Fmoc-Arg(Pbf)-Wang resin used for
the preparation of 4, the amount of Fmoc-Arg-OH pro-
duced did not change.
MW Fmoc SPPS was then continued to include the full
length NY-ESO-1 [154–180] sequence. Following chain
assembly and peptide cleavage with TFA–TIS–H2O–EDT
(94:1:2.5:2.5, v/v/v/v), the expected product 7 was ob-
served, together with a by-product 8 that lacked the
HMBA-Arg6-OH tail (Scheme 3 and Supporting Informa-
tion, figure 2). Several cleavage conditions were exam-
Other commercially available polystyrene resins such as
2-chlorotrityl resin and Rink resin were investigated for
Synthesis 2009, No. 20, 3460–3466 © Thieme Stuttgart · New York

3462
P. W. R. Harris, M. A. Brimble
PAPER
the preparation of the peptide containing the C-terminal could be detected in either sample, demonstrating that this
arginine tag. For both resins, Fmoc-Arg(Pbf)-HMBA- and other impurities had been eliminated. This result was
–
[Arg(Pbf)]6 was prepared on resin according to Scheme 2 especially apparent when comparing commercial Rink
and a representative sample was cleaved with TFA–TIS– Amide resin (Supporting Information, figure 3b) with the
H2O (95:2.5:2.5, v/v/v) for two hours (Supporting Infor- synthesized material (Supporting Information, figure 4a).
mation, figures 3a and 3b). For the synthesis using the 2- Performing the cleavage at 50 °C for five hours, however,
chlorotrityl resin, in addition to the expected product did result in the formation of two by-products (Supporting
Fmoc-Arg-HMBA-Arg6-OH (5), Fmoc-Arg-OH (6) and Information, figure 5), namely, Fmoc-Arg-OH (6) and
Fmoc-Arg7-OH (9) were also observed. For the synthesis Fmoc-Arg-HMBA-Arg-OH (18), demonstrating that care
using Rink Amide AM resin, the expected peptide, Fmoc- is required when handling the HMBA linker. For exam-
Arg-HMBA-Arg6-NH2 (10), was observed together with ple, pre-concentration of the TFA solution containing the
Fmoc-Arg-OH (6), AcO-HMBA-ArgnNH2 (n = 3,4,5,6) cleaved peptide should be avoided or the solution kept at
(12), Fmoc-Arg6-NH2 (13), AcNH-Arg6-NH2 (14) and 25 °C.
Fmoc-Arg-HMBA-OH (11). These products result from
Using the ‘in house’ Fmoc-Arg(Pbf)-HMBA-
incomplete coupling of either HMBA or Fmoc-Arg(Pbf)-
[Arg(Pbf)]6-HMPP resin and MW Fmoc SPPS, the 32-
OH or from incomplete esterification of the HMBA linker
residue polypeptide, NY-ESO-1 [155–180-HMBA-
by Fmoc-Arg(Pbf)-OH and the associated capping prod-
(Arg)6-OH] was obtained in excellent crude purity follow-
ing cleavage from the resin (Scheme 5 and Supporting In-
ucts.
The above observations suggested that the commercial formation, figure 6). LC-MS established that no peptide
resins were defective. We therefore prepared aminometh- material lacking the HMBA-Arg6-OH tag was detected,
yl polystyrene resin 15 as described20 with minor thus facilitating purification.
modifications21 to give a loading of 1.0 mmol/g by ele-
The formation of substantial amounts of by-product when
mental analysis. Condensation of Fmoc-Arg(Pbf)-
using commercial resins led us to postulate that erroneous
OCH2C6H4-OCH2CH2CO2H22 was quantitative by the
ninhydrin test after one hour using DIC as coupling re-
agent to give acid-cleavable HMPP (hydroxymethylphen-
O
O
C
O
C
O
NH-[Arg(Pbf)]6
oxypropionic acid) resin 17. For comparison, we also
prepared Rink Amide resin 16 by coupling the Rink
Linker9c with our ‘in house’ aminomethyl resin for two
hours using DIC.
O
Fmoc-Arg(Pbf)
1. MW Fmoc SPPS
2. TFA–TIS–EDT–H2O (94:1:2.5:2.5)
Using Fmoc SPPS protocols as described earlier
(Scheme 4), Fmoc-Arg(Pbf)-HMBA-[Arg(Pbf)]6- was
prepared on both resins and a small resin sample was
cleaved with TFA–TIS–H2O (95:2.5:2.5, v/v/v) for two
hours (Supporting Information, figure 4). The results ob-
tained using ‘in house’ resins were far superior to those
obtained using commercial resins.23 No Fmoc-Arg-OH
O
O
O
H2N
C-Arg6-CO2H
155–180
7
Scheme 5 MW Fmoc SPPS of NY-ESO-1 (155–180) coupled to an
arginine tag using resin prepared ‘in house’
OMe NHFmoc
prepared in-house
loading = 1.0 mmol/g
NH2
Fmoc-Arg(Pbf)O
MeO
OCH2CO2H
15
OCH2CH2CO2H
DIC, 1 h
DIC, 2 h
Rink Amide resin 16
Fmoc-Arg(Pbf)-HMPP resin 17
1. 20% piperidine, DMF, MW
2. Fmoc-Arg(Pbf)-OH
HBTU, DIPEA, DMF, MW
repeat
n times
n = 5 for HMPP resin
n = 6 for Rink Amide resin
1. 20% piperdine, DMF
2. HMBA, DIC, HOBt, 2 h
3. Fmoc-Arg(Pbf)-OH
DIC, DMAP, 1 h (repeat 2 x)
4 Ac2O, DMAP, 30 min
5. TFA, TIS, H2O, 2 h
Fmoc-Arg-HMBA-Arg6NH2 10
Fmoc-Arg-HMBA-Arg6OH 5
Scheme 4 Synthesis of Fmoc-Arg-HMBA-Arg6 using SPPS on resins prepared ‘in house’
Synthesis 2009, No. 20, 3460–3466 © Thieme Stuttgart · New York

PAPER
Synthesis of a [Cys155–Arg180] Fragment of NY-ESO-1
3463
ually. Briefly, manual synthesis included: a) Fmoc deprotection
with 20% piperidine for 5 min, then 10 min washing with DMF
(5×); b) coupling of the Fmoc amino acid (5 equiv) in the presence
of 0.5 M HBTU in DMF (4.9 equiv) and 2 M DIPEA in NMP (10
equiv) for 45 min and washing with DMF (5×). The progress of the
acylation was monitored by the ninhydrin test. A minimum amount
of DMF was used for dissolution of the Fmoc amino acid.
nucleophilic functionalities such as hydroxyl (or phenol-
ic) groups were present and that, during acylation of the
resin-bound N-terminal amino group with HMBA, these
functionalities may also react. Subsequent SPPS cycles
lead to the undesired peptide products.
In order to negate the problems caused by these function-
alities, we reacted commercial Fmoc-Arg(Pbf)-Wang res-
in with either Ac2O/DMAP, Bz2O/DMAP or HMBA/
DIC/HOBt followed by Ac2O/DMAP. These pre-capped
resins were then reacted according to Scheme 2 to give
resin-bound Fmoc-Arg(Pbf)-HMBA-Arg(PBf)6-Wang
resin. Cleavage with TFA–TIS–H2O (95:2.5:2.5, v/v/v)
for two hours, in all cases, resulted in the formation of
Analytical HPLC and LC-MS were performed using a Dionex Ulti-
mate 3000 equipped with a four-channel UV detector using a Gem-
ini C18 (5mm; 2.1 × 50 mm) column (Phenomenex) at 0.2 mL/min
using a linear gradient of 5→65%B over 21 min (ca. 3%B per
minute). The solvent system used was A (0.1% TFA in H2O) and B
(0.1% TFA in MeCN) with detection at 210, 230, 254 and 280 nm.
Ratios of products were determined by integration of spectra re-
corded at 210 nm. Peptide masses were confirmed by an inline
Fmoc-Arg-OH (6), although in less than the 20% obtained Thermo Finnigan MSQ mass spectrometer using ESI in the positive
mode. Where appropriate, a post column addition of propionic
originally when no capping step was performed (7%
acid–2-propanol (3:1) was used to enhance ionization.25 Peptides
Fmoc-Arg for the resin capped with Ac2O/DMAP, 10%
were purified using either a Waters 600E system or a Gilson 215
for the resin capped with Bz2O/DMAP or HMBA/DIC/
system using a Waters Xterra preparative column (10 mm, 19 × 250
HOBt followed by Ac2O/DMAP). These findings demon-
mm) using a flow rate of 10 mL/min and eluted with an appropriate
strate that either there are other reactive functional groups
not acylated by Ac2O or these groups are subsequently un-
masked by the conditions used during Fmoc SPPS, which
lead to undesired products.
shallow gradient of increasing concentration of MeCN containing
0.1% TFA. Fractions were collected, analysed by either HPLC or
LC-MS, pooled and lyophilised.
Attenuated total reflection infrared (ATR-IR) spectroscopy was re-
corded on a Bruker Alpha spectrometer.
For the solid-phase synthesis of peptides using the Fmoc
method it is therefore recommended that resins are pre-
pared ‘in house’. If commercial resins are used, it is rec-
ommended that a capping step be included at the onset of
the synthesis. In spite of the difficulties experienced here-
in, the synthesis of the extremely hydrophobic C-terminal
region of the NY-ESO-1 protein was successful. Further
work will focus on using this fragment in a native chemi-
cal ligation reaction to prepare the biologically important
NY-ESO-1 cancer protein.
Preparation of Resin-Bound Fmoc-Arg(Pbf)-HMBA-
Arg(Pbf)6; General Procedure
Using commercial Fmoc-Arg(Pbf)-Wang resin (loading = 0.47
mmol/g) on a 0.1 mmol scale. Fmoc SPPS was performed in a CEM
Liberty microwave peptide synthesizer as described previously, to
afford Fmoc-Arg(Pbf)6-Wang resin. This resin (6 mg) was shaken
with TFA–TIS–H2O (95:2.5:2.5, v/v/v; 0.2 mL) for 2 h. 0.1 mL of
this was removed and 0.9 mL of cold Et2O was added, resulting in
a precipitate. Following storage at 0 °C for 20 min, the solid was
isolated by centrifugation, re-dissolved in H2O–MeCN (1:1) con-
taining 0.1% TFA (0.1 mL), and analysed by LC-MS.
All solvents and reagents were used as supplied. O-(Benzotriazol-
1-yl)-N,N,N¢,N¢¢-tetramethyluronium hexafluorophosphate (HB-
TU), N-hydroxybenzotriazole (HOBt) were purchased from Ad-
vanced Chemtech (Louisville, KY). DMF (AR grade) and MeCN
(HPLC grade) were purchased from Ajax Chemicals, methane-
sulfonic acid (MsOH), N-(hydroxymethyl)phthalimide, diisopropy-
lethylamine (DIPEA), piperidine, ethanedithiol (EDT), and
triisopropylsilane (TIS) were purchased from Aldrich and N-meth-
ylpyrrolidine (NMP) was purchased from Fluka. TFA was pur-
chased from Scharlau. The Rink linker and HMBA linker were
purchased from GL Biochem. Fmoc-Arg(Pbf)-OCH2-C6H4-
OCH2CH2CO2H was purchased from NeoSystem. Copoly(styrene-
1%-divinylbenzene) resin (Bio beads S-X1; 200–400 mesh) was
purchased from Bio-Rad.
Fmoc-Arg(Pbf)6-OH (19)
tR = 12.2 min.
MS: m/z [M + 2H]2+ calcd: 589.6; found: 589.6.
MS: m/z [M + 3H]3+ calcd: 393.4; found: 393.1.
The remainder of the resin material was subjected to N-terminal
Fmoc removal as described in the general procedure. HMBA (46
mg, 0.3 mmol) and HOBt (92 mg, 0.6 mmol) were dissolved in
DMF (2 mL), DIC (61 mL, 0.4 equiv) was added and the solution
was added to the resin. The mixture was shaken for 2 h after which
time a negative ninhydrin test was observed. The resin was then
washed with DMF (5×), then CH2Cl2 (5×) and dried under nitrogen.
This resin (6 mg) was shaken with TFA–TIS–H2O (95:2.5:2.5,
v/v/v; 0.2 mL) for 2 h. 0.1 mL was then removed and 0.9 mL of cold
Et2O was added resulting in a precipitate. Following storage at 0 °C
for 20 min, the solid was isolated by centrifugation, re-dissolved in
H2O–MeCN (1:1) containing 0.1% TFA (0.1 mL), and analysed by
LC-MS.
Fmoc-amino acids were purchased from CEM or GL Biochem with
the following side chain protection: Fmoc-Arg(Pbf)-OH, Fmoc-
Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-
Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Ser(OtBu)-OH, Fmoc-Thr(OtBu)-OH and Fmoc-Trp(Boc)-OH.
Fmoc Arg(Pbf) attached to Wang polystyrene resin (loading = 0.47
mmol/g), H2N-Arg(Pbf) attached to 2-chlorotrityl resin
(loading = 0.59 mmol/g) and Fmoc-Rink Amide AM resin
(loading = 0.6 mmol/g) were purchased from either Varian Inc.,
Peptides International or GL Biochem.
HO-HMBA-Arg(Pbf)6-OH (20)
tR = 1 min.
MS: m/z [M + 2H]2+ calcd: 545.6; found:545.3.
To the resin was then added a solution of Fmoc-Arg(Pbf)-OH
(0.256 g, 0.4 mmol) in DMF (2 mL) and DIC (76 mL, 0.5 mmol) fol-
lowed by the dropwise addition of DMAP (1.22 mg, 0.01 mmol) in
DMF (0.122 mL) over 1 min. The suspension was shaken gently for
1 h, drained, and the acylation repeated. The resin was washed with
Fmoc solid-phase peptide synthesis was performed using a Liberty
Microwave Peptide Synthesiser (CEM Corporation, Mathews, NC)
either using the Fmoc–tBu strategy as outlined previously24 or man-
Synthesis 2009, No. 20, 3460–3466 © Thieme Stuttgart · New York

3464
P. W. R. Harris, M. A. Brimble
PAPER
DMF (5×) then made mobile in the minimum amount of DMF and
Ac2O (95 mL, 1 mmol) and DMAP (1.22 mg, 0.01 mmol) in DMF
(0.122 mL) were added and the mixture shaken for 30 min. The res-
in was then washed with DMF (5×), then CH2Cl2 (5×) and dried un-
der nitrogen. The resin (8 mg) was shaken with TFA–TIS–H2O
(95:2.5:2.5, v/v/v; 0.2 mL) for 2 h. 0.1 mL of this was removed and
0.9 mL of cold Et2O was added resulting in a precipitate. Following
storage at 0 °C for 20 min, the solid was isolated by centrifugation,
re-dissolved in H2O–MeCN (1:1) containing 0.1% TFA (0.1 mL),
and analysed by LC-MS.
ESI-MS: m/z [M + 1H]1+ calcd: 397.2; found: 397.0.
Preparation of Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6-NH2 on
Commercial Fmoc-Rink Amide AM Resin
The general procedure as outlined above was followed to afford res-
in-bound Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6-NH-resin. The resin
(10 mg) was cleaved with TFA–TIS–H2O (95:2.5:2.5, v/v/v; 0.2
mL) for 2 h. 0.1 mL of this was removed and 0.9 mL of cold Et2O
was added, resulting in a precipitate. Following storage at 0 °C for
20 min, the solid was isolated by centrifugation, re-dissolved in
H2O–MeCN (1:1) containing 0.1% TFA (0.1 mL), and analysed by
LC-MS.
Fmoc-Arg-HMBA-Arg6-OH (5)
Yield: 80%; tR = 13.6 min.
AcO-HMBA-ArgnNH2 (n = 3,4,5,6) (12)
Yield: 23%; tR = 9.4 (23%).
MS: m/z [M + 2H]2+ calcd: 734.7; found: 734.6.
MS: m/z [M + 3H]3+ calcd: 490.1; found: 490.0.
Fmoc-Arg-OH (6)
MS: m/z [M(n = 3) + 2H]2+ calcd: 332.3; found: 331.5.
MS: m/z [M(n = 4) + 2H]2+ calcd: 410.4; found: 409.7.
MS: m/z [M(n = 5) + 2H]2+ calcd: 488.5; found: 487.8.
MS: m/z [M(n = 6) + 2H]2+ calcd: 566.6; found: 566.0.
Fmoc-Arg6-NH2 (13)
Yield: 20%; tR = 16.2 min.
MS: m/z [M + 1H]1+ calcd: 397.2; found: 397.0.
Preparation of NY-ESO-1 154–180 with an Arginine Tag on
Commercial Fmoc-Arg(Pbf) Wang Resin
Yield: 24%; tR = 12.2.
Using Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6 on Wang resin, the pep-
tide was synthesised using MW Fmoc SPPS on a 0.1 mmol scale.
Following chain assembly, the resin was cleaved with TFA–TIS–
EDT–H2O (94:1:2.5:2.5, v/v/v; 10 mL) for 2 h. The resin was
drained and the filtrate was precipitated by the addition of cold Et2O
(100 mL) and isolated by centrifugation. The solid was dissolved in
H2O–MeCN (1:1) containing 0.1% TFA (0.1 mL) and lyophilised
to afford 184 mg of crude peptide. Purification by preparative
HPLC gave 7 (24.4 mg, 5.7%).
MS: m/z [M + 2H]2+ calcd: 589.6; found: 589.1.
Fmoc-Arg-HMBA-Arg6-NH2 (10)
Yield: 38%; tR = 13.0.
MS: m/z [M + 2H]2+ calcd: 489.3; found: 489.9.
AcNH-Arg6-NH2 (14)
Yield: 5.2%; tR = 14.2.
MS: m/z [M + 2H]2+ calcd: 499.6; found: 499.0.
NY-ESO-1 [NH2-154-180-HMBA-Arg6-OH] (7)
Yield: 80%; tR = 19.6 min.
MS: m/z [M + 4H]4+ calcd 1061.8; found: 1062.8.
MS: m/z [M + 5H]5+ calcd: 849.6; found: 849.7.
Fmoc-Arg-OH (6)
Yield: 4%; tR = 15.2.
MS: m/z [M + 1H]1+ calcd: 397.2; found: 397.0.
Fmoc-Arg-HMBA-NH2
NY-ESO-1 [H2N-154-180-OH] (8)
Yield: 20%; tR = 21.6 min.
MS: m/z [M + 3H]3+ calcd: 1058.3; found: 1058.1.
Yield: 6%; tR = 17.0.
MS: m/z [M + 1H]1+ calcd: 530.3; found: 530.2.
Preparation of Aminomethyl Polysytrene Resin 15
MS: m/z [M + CO2 + 3H]3+ calcd: 1073.0; found: 1072.9.
To a stirred suspension of Biobeads SX-1 (5 g) in CH2Cl2 (90 mL),
was added N-(hydroxymethyl)phthalimide (0.974 g, 5.5 mmol) and
MsOH (10 mL) and the mixture was stirred at r.t. for 5 h. The resin
was filtered and washed successively with CH2Cl2 (4 × 150 mL)
and EtOH (4 × 150 mL) and dried under vacuum to afford 6.06 g of
resin. ATR-IR of a resin sample showed a peak at 1715 cm–1 indi-
cating incorporation of the phthalimide group. The resin was then
suspended in 5% v/v hydrazine hydrate in EtOH (40 mL) and the
mixture was refluxed for 5 h. The hot mixture was filtered immedi-
ately, washed successively with hot EtOH (400 mL), hot MeOH
(400 mL), DMF (200 mL), CH2Cl2 (200 mL), CH2Cl2–TFA (100
mL, 1:1, v/v), CH2Cl2 (200 mL), 10% DIPEA in DMF (200 mL),
DMF (200 mL), CH2Cl2 (200 mL) and dried under vacuum, to give
3.1 g of 15 which contained 1.39% N by elemental analysis, corre-
sponding to a loading of 0.99 mmol/g. ATR-IR of a resin sample
showed the absence of carbonyl peaks.
Preparation of Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6-OH on
Commercial H2N-Fmoc-Arg(Pbf)-2-Cl-trityl Resin
The general procedure as outlined above was followed to afford res-
in-bound Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6-O-resin. The resin (10
mg) was cleaved with TFA–TIS–H2O (95:2.5:2.5, v/v/v; 0.2 mL)
for 2 h. 0.1 mL of this was removed and 0.9 mL of cold Et2O was
added, resulting in a precipitate. Following storage at 0 °C for 20
min, the solid was isolated by centrifugation, re-dissolved in H2O–
MeCN (1:1) containing 0.1% TFA (0.1 mL), and analysed by LC-
MS.
Fmoc-Arg7-OH (9)
Yield: 19%; tR = 12.1 min.
ESI-MS: m/z [M + 2H]2+ calcd: 667.7; found: 668.1, m/z [M + 3H]3+
calcd: 445.5found: 445.3.
Fmoc-Arg-HMBA-Arg6-OH (5)
Yield: 74%; tR = 12.9 min.
ESI-MS: m/z [M + 2H]2+ calcd: 734.7; found: 735.1, m/z [M + 3H]3+
Preparation of Rink Amide Resin 16
Aminomethyl PS resin 15 with a loading of 0.99 mmol/g (0.3 g, 0.3
mmol) was swelled in DMF for 5 min. 4-[(2,4-Dimethoxyphe-
nyl)(fmoc-amino)methyl]phenoxyacetic acid (Rink Linker) (0.485
g, 0.9 mmol) and HOBt (0.138 g, 0.9 mmol) were dissolved in DMF
(3 mL), DIC (0.138 mL, 0.9 mmol) was added and the solution was
allowed to stand for 5 min. The activated linker was then added to
calcd: 490.1; found: 490.1.
Fmoc-Arg-OH (6)
Yield: 7%; tR = 15.8 min.
Synthesis 2009, No. 20, 3460–3466 © Thieme Stuttgart · New York

PAPER
Synthesis of a [Cys155–Arg180] Fragment of NY-ESO-1
3465
the resin and the mixture was shaken for 2 h, after which a negative
ninhydrin test was obtained. The resin was drained, washed with
DMF (5×), washed with CH2Cl2 (5×) and dried in vacuo. The load-
ing was determined to be 0.81 mmol/g by the Fmoc-release assay.
Yield: 22%; tR = 15.5 min.
MS: m/z [M + 2H]2+ calcd: 687.5; found: 687.5.
MS: m/z [M + 3H]3+ calcd: 344.3; found: 344.3.
Fmoc-Arg-OH (6)
Preparation of HMPP Resin 17
Yield: 13%; tR = 15.9 min.
MS: m/z [M + 1H]1+ calcd: 397.2; found: 397.0.
Aminomethyl PS resin 15 with a loading of 0.99 mmol/g (0.10 g,
0.1 mmol) was swelled in DMF for 5 min. 4-[Fmoc-(NG-Pbf)-argi-
nyloxymethyl]phenoxypropionic acid (0.166 g, 0.2 mmol) was dis-
solved in CH2Cl2 (2 mL), DIC (31 mL, 0.2 mmol) was added and the
solution was allowed to stand for 5 min. The activated linker was
then added to the resin and the mixture was shaken for 1 h, after
which a negative ninhydrin test was obtained. The resin was
drained, washed with DMF (5×), washed with CH2Cl2 (5×) and
dried in vacuo. The loading was determined to be 0.55 mmol/g by
the Fmoc release assay.
Preparation of NY-ESO-1 154-180 with an Arginine Tag
Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6 on HMPP resin was synthesised
as described above on a 0.1 mmol scale and chain-elongation was
performed using MW Fmoc SPPS. Following chain assembly, the
resin was cleaved with TFA–TIS–EDT–H2O (94:1:2.5:2.5, v/v/v;
10 mL) for 2 h. The resin was drained and the filtrate was precipi-
tated by the addition of cold Et2O (100 mL) and isolated by centrif-
ugation. The solid was dissolved in H2O–MeCN (1:1) containing
0.1% TFA and lyophilised to afford 154 mg of crude 7.
Preparation of Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6 on Rink
Amide Resin
LC-MS: tR = 19.6 min.
The general procedure outlined above was followed, however, all
couplings were performed manually. Following completion of the
solid-phase synthesis, resin-bound Fmoc-Arg(Pbf)-HMBA-
Arg(Pbf)6-NH-resin was obtained. The resin (10 mg) was cleaved
with TFA–TIS–H2O (95:2.5:2.5, v/v/v; 0.2 mL) for 2 h. 0.1 mL of
this was removed and 0.9 mL of cold Et2O was added, resulting in
a precipitate. Following storage at 0 °C for 20 min, the solid was
isolated by centrifugation, re-dissolved in H2O–MeCN (1:1) con-
taining 0.1% TFA (0.1 mL), and analysed by LC-MS.
MS: m/z [M + 3H]3+ calcd: 1415.4; found: 1415.8.
MS: m/z [M + 4H]4+ calcd: 1061.8; found: 1062.1.
MS: m/z [M + 5H]5+ calcd: 849.6; found: 849.8.
Purification by preparative HPLC gave 7 (27.12 mg, 6.4%).
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
AcOCH2PhCONH-Arg6-NH2 (12)
Yield: 11%; tR = 9.2 min.
MS: m/z [M + 2H]2+ calcd: 566.6; found: 566.0.
Acknowledgment
The authors wish to thank The Maurice Wilkins Centre for Molecu-
lar Biodiscovery for financial support and Professor Stephen Kent
for stimulating discussions.
Fmoc-Arg-HMBA-Arg6-NH2 (10)
Yield: 89%; tR = 13.2 min.
MS: m/z [M + 2H]2+ calcd: 734.2; found: 734.3.
MS: m/z [M + 3H]3+ calcd: 489.8; found: 489.8.
References
(1) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35,
161..
(2) Bouillon, I.; Sourai, M.; Miller, M. K.; Krchnak, V. J. Comb.
Chem. 2009, 11, 213.
(3) Pugh, K. C.; York, E. J.; Stewart, J. M. Int. J. Pept. Protein
Res. 1992, 40, 208.
(4) Wang, S.-S. J. Am. Chem. Soc. 1973, 95, 1328.
(5) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J.
Pept. Protein Res. 1991, 37, 513.
(6) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
(7) Atherton, E.; Benoiton, N. L.; Brown, E.; Sheppard, R. C.;
Williams, B. J. J. Chem. Soc., Chem. Commun. 1981, 336.
(8) Atherton, E.; Logan, C. J.; Sheppard, R. C. J. Chem. Soc.,
Perkin Trans. 1 1981, 538.
(9) (a) For Wang linker equivalents, see: Zinieris, N.; Kokinaki,
S.; Leondiadis, L.; Ferderigos, N. Synlett 2006, 2789.
(b) For 2-Cl-trityl linker equivalents, see: Zikos, C.;
Livaniuo, E.; Leondiadis, L.; Ferderigos, N.; Ithakissios, D.
S.; Evangelatos, G. P. J. Pept. Sci. 2005, 8, 419. (c) For
Rink Amide linker equivalents, see: Bertnatowicz, M. S.;
Daniels, S. B.; Koster, H. Tetrahedron Lett. 1989, 30, 4645.
(10) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776.
Preparation of Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6 on HMPP
Resin
The general procedure as outlined above was followed to afford res-
in-bound Fmoc-Arg(Pbf)-HMBA-Arg(Pbf)6-O-resin. This resin
(10 mg) was cleaved with TFA–TIS–H2O (95:2.5:2.5, v/v/v; 0.2
mL) for 2 h. 0.1 mL of this was removed and 0.9 mL of cold Et2O
was added, resulting in a precipitate. Following storage at 0 °C for
20 min, the solid was isolated by centrifugation, re-dissolved in
H2O–MeCN (1:1) containing 0.1% TFA (0.1 mL), and analysed by
LC-MS.
Fmoc-Arg-HMBA-Arg6-OH (5)
tR = 12.8 min.
ESI-MS: m/z [M + 2H]2+ calcd: 734.7; found: 734.9.
ESI-MS: m/z [M + 3H]3+ calcd: 490.1; found: 490.2.
The remaining cleavage solution (0.1 mL) was heated to 50 °C in an
Eppendorf with shaking for 5 h. The mixture was cooled to r.t. and
cold Et2O (0.9 mL) was added, resulting in a precipitate. Following
storage at 0 °C for 20 min, the solid was isolated by centrifugation,
re-dissolved in H2O–MeCN (1:1) containing 0.1% TFA (0.1 mL),
and analysed by LC-MS.
Fmoc-Arg-HMBA-Arg6-OH (5)
Yield: 65%; tR = 13.0 min.
MS: m/z [M + 3H]3+ calcd: 490.1; found: 490.2.
(11) Harris, P. W. R.; Brimble, M. A. manuscript in preparation.
(12) (a) Atherton, E.; Logan, C. J.; Sheppard, R. C. J. Chem. Soc.,
Perkin Trans. 1 1981, 529. (b) Choma, C. T.; Robillard, G.
T.; Englebretsen, D. R. Tetrahedron Lett. 1998, 39, 2417.
Fmoc-Arg-HMBA-Arg-OH (18)
Synthesis 2009, No. 20, 3460–3466 © Thieme Stuttgart · New York

3466
P. W. R. Harris, M. A. Brimble
PAPER
(19) Sole, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399.
(20) Mitchell, A. R.; Kent, S. B. H.; Englenhard, M.; Merrifield,
R. B. J. Org. Chem. 1978, 43, 2845.
(13) Mellor, S. L.; Wellings, D. A.; Fehrentz, J.-A.; Paris, M.;
Martinez, J.; Ede, N. J.; Bray, A. M.; Evans, D. J.;
Bloomberg, G. B. In Fmoc solid phase peptide synthesis: a
practical approach; Oxford University Press: Oxford, 2000,
145–149.
(14) Rittel, W. R. Helv. Chim. Acta 1962, 45, 465.
(15) Reaction of Fmoc-Arg(Pbf)-OH with TFA–TIS–H2O
(95:2.5:2.5, v/v/v) gave identical HPLC and mass spectra to
6.
(16) Sheppard et al. reported hydrolysis at the HMBA linker
when attempting ammonolysis. This was determined to be
due to moisture present in the resin, see: Brown, E.;
Sheppard, R. C.; Williams, B. J. J. Chem. Soc., Perkin Trans.
1 1983, 75.
(21) Kent, S. B. H. personal communication.
(22) (a) Albericio, F.; Barany, G. Int. J. Pept. Protein Res. 1985,
26, 92. (b) NeoMPS S.A. International Patent Application
W02005/095332, 2005.
(23) According to reference 2, resins that swell less than 7 mL/g
in CH2Cl2 should be rejected. For commercially obtained
Wang, Rink Amide AM, and 2-Cl trityl reins used here, the
swelling was 6.1, 5 and 4.5 mL/g, respectively, while for the
‘in house’ reins 16 and 17, the swelling was 9 and 9.7 mL/g,
respectively.
(24) Harris, P. W. R.; Williams, G. M.; Shepherd, P.; Brimble, M.
(17) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept. Protein
Res. 1999, 36, 255.
(18) Reagent, R.; Albericio, F.; Kenib-Cordonier, N.;
Bianaclanan, S.; Gera, L.; Masada, R. I.; Hudson, D.;
Barany, G. J. Org. Chem. 1990, 55, 3730.
A. Int. J. Pept. Res. Ther. 2008, 14, 387.
(25) Apffel, A.; Fischer, S.; Goldberg, G.; Goodley, P. C.;
Kuhlmann, F. E. J. Chromatogr., A 1995, 712, 177.
Synthesis 2009, No. 20, 3460–3466 © Thieme Stuttgart · New York