Synthesis and biological evaluation of two chemically modified peptide epitopes for the class i MHC protein HLA-B*2705

Article abstract of DOI:10.1039/b611170j

The T-cell receptor of a CD8⁺ T-cell recognises peptide epitopes bound by class I major histocompatibility complex (MHC) glycoproteins presented in a groove on their upper surface. Within the groove of the MHC molecule are 6 pockets, two of whi

Full text of DOI:10.1039/b611170j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of two chemically modified peptide
epitopes for the class I MHC protein HLA-B*2705
Matthew A. Jones,a Andrew D. Hislopb and John S. Snaith*a
Received 2nd August 2006, Accepted 23rd August 2006
First published as an Advance Article on the web 8th September 2006
DOI: 10.1039/b611170j
The T-cell receptor of a CD8+ T-cell recognises peptide epitopes bound by class I major
histocompatibility complex (MHC) glycoproteins presented in a groove on their upper surface. Within
the groove of the MHC molecule are 6 pockets, two of which mostly display a high degree of specificity
for binding amino acids capable of making conserved and energetically favourable contacts with the
MHC. One type of MHC molecule, HLA-B*2705, preferentially binds peptides containing an arginine
at position 2. In an effort to increase the affinity of peptides for HLA-B*2705, potentially leading to
better immune responses to such a peptide, we synthesised two modified epitopes where the amino acid
at position 2 involved in anchoring the peptide to the class I molecule was replaced with the
a-methylated b,c-unsaturated arginine analogue 2-(S)-amino-5-guanidino-2-methyl-pent-3-enoic acid.
The latter was prepared via a multi-step synthetic sequence, starting from a-methyl serine, and
incorporated into dipeptides which were fragment-coupled to resin-bound heptameric peptides yielding
the target nonameric sequences. Biological characterisation indicated that the modified peptides were
poorer than the native peptides at stabilising empty class I MHC complexes, and cells sensitised with
these peptides were not recognised as well by cognate CD8+ T-cells, where available, compared to those
sensitised with the native peptide. We suggest that the modifications made to the peptide have decreased
its ability to bind to the peptide binding groove of HLA-B*2705 molecules which may explain the
decrease in recognition by cytotoxic T-cells when compared to the native peptide.
act by increasing the affinity of the epitope for an MHC molecule,
Introduction
by increasing the affinity of the peptide–MHC complex for the
Our immune system protects us against infectious agents and
T-cell receptor, or by stimulating a broader T-cell response against
malignancies through the recognition of specific antigens derived
the pathogen. The approach which has been most widely adopted,
from pathogens. Specific cell-mediated immune responses involve
and which has been shown to produce significantly improved
T-lymphocytes that respond to peptide epitopes, typically of
vaccines, is to increase the affinity of the epitope for the MHC
between 8 and 20 amino acids in length, derived from these agents.1
The peptides are recognised by CD8+ T-cells only when bound to
cell-surface protein receptors known as major histocompatibility
complex (MHC) molecules. Our particular interest is in class I
MHC molecules, which are on the surface of all nucleated cells
and present peptides of 8–10 amino acids in length, derived
mostly from intracellular proteins. Class I MHC molecules show
a strong preference for peptides with particular residues—termed
anchor residues—in certain positions along the chain. X-Ray
crystallographic evidence has shown that the peptides bind in a
long groove in the protein, with the anchor residues fitting into
specific pockets in the binding groove.2
Recent advances in our understanding of the cellular immune
response at the molecular level have led to a new strategy
for vaccine development. The strategy involves the chemical
modification of viral epitopes to make them more immunogenic, a
process termed epitope enhancement.3 Epitope enhancement can
molecule.4 To increase the peptide affinity for MHC molecules,
one can take advantage of known sequence motifs for peptide
binding5 and modify the anchor residues that provide much of the
specificity of binding to the MHC molecule.
The human allele HLA-B*2705 is one of the most extensively
studied class I MHC proteins, with a very clear motif for binding
peptides. Its association with a variety of autoimmune diseases
meant that HLA-B*2705–peptide complexes were amongst the
first to be crystallised. HLA-B*2705 has a high specificity for
peptides of 9 residues in length with an arginine at position 2, and
analysis of the X-ray structure has shown that the side chain of
this arginine (Arg 412 in crystallographic numbering) is bound in
a polar pocket in the protein formed by His 9, Thr 24, Glu 45 and
Cys 67 (Fig. 1).2
To increase the affinity of epitopes for HLA-B*2705, we
designed an arginine isostere containing a double bond to restrict
the side-chain flexibility.7,8 We envisaged that by introducing a
conformational restriction within the P2 anchor residue we could
reduce the entropic cost of binding a peptide to HLA-B*2705,
thereby enhancing the peptide–MHC affinity and possibly, in
a vaccination setting, leading to a stronger immune response.9
We chose two peptide epitopes on which to test this hypothesis:
aSchool of Chemistry, University of Birmingham, Birmingham, UK B15
2TT. E-mail: j.s.snaith@bham.ac.uk; Fax: +44 121 414 4403; Tel: +44 121
414 4363
bCancer Research UK Institute for Cancer Studies, University of Birming-
ham, Birmingham, UK B15 2TT
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compound have been reported,15 none appeared to be amenable to
scale-up, and so we decided to explore a route involving resolution
of the commercially available ( )-a-methylserine 4. The racemic
amino acid is inexpensive, and we believed that resolution by
diastereomeric salt formation with camphorsulfonic acid should
be possible.
Esterification of ( )-a-methyl-serine 4 was achieved using
methanol and thionyl chloride16 (Scheme 1), and the resulting
hydrochloride salt was exchanged for the camphorsulfonic acid
salt by heating with (+)-camphorsulfonic acid in CH2Cl2 and
acetone. Crystallisation overnight from the same solvent mixture
gave an excellent recovery of the a-methylserine methyl ester–(+)-
camphorsulfonic acid salt, enriched in the (S)-enantiomer of the
amino ester. The crystals were redissolved in CH2Cl2 and acetone
and the fractional crystallisation procedure was repeated. The
enantiomerically enriched amino ester was isolated by carbamate
protection with (Boc)2O, affording 3 in 34% overall yield from
a-methylserine methyl ester, i.e. 68% based on recovery of a single
enantiomer. The resolution was routinely performed on a 20 g
scale.
Fig. 1 Interactions of Arg 412 with residues in the binding pocket. Figure
prepared using Rasmol6 with data from Madden et al.2
GRAFVTIGK, residues 314–322 of the HIV envelope protein
gp120;10 and RRIYDLIEL, residues 258–266 of the Epstein-Barr
virus protein EBNA3C.11
Design
Scheme 1 Reagents and conditions: i. SOCl2, MeOH, D, 16 h, 95%; ii.
(+)-CSA, D, CH2Cl2, acetone; iii. fractional crystallisation; iv. (Boc)2O,
Et3N, CHCl3, D, 16 h, 34% over 3 steps, 68% based on recovery of single
enantiomer; v. PCC, CH2Cl2, 16 h, 54%; vi. Ph3PCHCHO, C6H6, 4 days,
70%; vii. NaBH4, 0.4 M CeCl3, MeOH, 50%.
Inspection of the published crystal structures of HLA-B*2705
containing bound peptides2,12 showed that the P2 arginine was
bound in an extended conformation, making the analogue 1
containing a double bond essentially isosteric (Fig. 1 and 2).
Although there are a number of reported syntheses of amino acids
containing b,c-unsaturation,13 they are prone to isomerisation
of the double bond into conjugation with the carbonyl group,
making their incorporation into peptides very challenging.14 To
circumvent this problem we decided to introduce an a-methyl
substituent,14 making our target the amino acid 2. As our starting
point for the synthesis we chose the known orthogonally protected
amino acid (S)-N(Boc)-a-methylserine methyl ester 3, which we
reasoned could be elaborated towards the target b,c-unsaturated
isostere by oxidation to the aldehyde and subsequent Wittig
chemistry.
The enantiopurity of the (S)-N(Boc)-a-methylserine methyl
ester 3 was determined by formation of the Mosher’s ester.17
Examination of the 1H NMR revealed a dr of >70 : 1, and
the absolute stereochemistry of the product was confirmed as
S by comparison of the specific optical rotation with literature
values.18 As the specific rotation of 3 at 589 nm was so small, we
also measured it at a range of wavelengths to provide additional
reference data (see Experimental section).
With multigram quantities of the enantiomerically enriched pre-
cursor 3 available, we set out to install the unsaturated side-chain
using Wittig chemistry (Scheme 1). PCC oxidation of alcohol 3
under standard conditions proceeded smoothly to afford aldehyde
5 in a 54% yield, and this was followed by a Wittig reaction with the
commercially available (formylmethylene)triphenyl phosphorane,
giving the alkene 6 in 70% yield, exclusively as the E-stereoisomer.
Diisobutylaluminium hydride reduction of 6 proceeded smoothly
on a small scale, but the yields were not reproducible on a larger
scale. Switching to the Luche reduction furnished the required
allylic alcohol 7 in 58% yield, and this procedure could be reliably
scaled to several grams.19
Fig. 2 P2 arginine, analogue 1 and target amino acid 2.
The next step was to introduce the guanidino function required
for the arginine isostere. Since guanidinylation of an amine is one
of the most straightforward methods for the introduction of the
guanidino group,20 we set about the synthesis of the triprotected
ornithine analogue 8 (Scheme 2). Mesylation of alcohol 7, followed
by treatment with sodium azide gave the corresponding azide in
Results and discussion
Our synthetic route required a significant quantity of (S)-N(Boc)-
a-methylserine methyl ester 3. Although several syntheses of this
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There are no reported examples of arginine acid fluorides, and
so we attempted to prepare the acid fluoride of tri-Boc-protected
arginine. Our synthesis started with the commercially available
(S)-a-N(Boc)-d-N(Cbz)-ornithine 12. Hydrogenolysis to remove
the Cbz group was followed by guanidinylation using bis(tert-
butoxycarbonyl)triflylguanidine23 to form tri-Boc-protected argi-
nine 13 in 76% yield after chromatography (Scheme 4). Unfor-
tunately, treatment with cyanuric fluoride under the reported
conditions21 did not yield any of the desired acid fluoride, with
only unreacted starting material recovered.
Scheme 2 Reagents and conditions: i. MsCl, Et3N, CH2Cl2, 16 h; ii.
NaN3, CH3CN, D, 16 h, 75% over 2 steps; iii. Ph3P, THF–H2O, D, 2 h,
20%; iv. 28% aq. NH3, CH2Cl2, 4 h; v. FmocOSu, NaHCO3, acetone–H2O,
16 h, 25% for 3 steps.
75% yield over two steps, but the Staudinger reduction to give
amine 9 was low-yielding (20%), and separation of the amine
from residual triphenylphosphine oxide was difficult, prompting
us to explore an alternative approach. Instead, treatment of the
mesylate with aqueous ammonia gave the desired amine 9, which
was Fmoc-protected to afford 8 in 25% yield over 3 steps.
We were concerned that the incorporation of a sterically
congested a-methylated amino acid into a peptide could be low-
yielding, and that this steric crowding could also affect subsequent
peptide coupling steps. Since the quaternary stereogenic centre is
not susceptible to racemisation, we elected to employ a fragment
coupling, first coupling our isostere at the N-terminus to form
a dipeptide, and then attaching this to a heptameric sequence on
resin to form the target nonameric peptide. To minimise protecting
group manipulations, we decided to couple the protected ornithine
analogue 8 to give a dipeptide, and then guanidinylate the d-amino
group to form the arginine isostere.
Scheme 4 Reagents and conditions: i. H2, Pd–C, MeOH, 16 h, 99%;
ii. Bis(tert-butoxycarbonyl)triflylguanidine, MeOH, 3 days, 76%; iii. 10,
iPr2EtN, HATU, HOAt, 16 h, 13%.
Two dipeptides were required to make our target nonapeptides,
one incorporating glycine and the other incorporating arginine.
Synthesis of the first dipeptide was straightforward. Boc group
removal from 8 with TFA, giving 10, was followed by reaction with
the acid fluoride of N-Boc-glycine21 to give the coupled product
11 in 66% yield over two steps (Scheme 3). A range of amino acid
fluorides have been reported, and they have found application as
highly activated coupling partners in peptide bond formation.22
Our failure to prepare the protected arginine fluoride led us to
turn to the coupling of 10 and 13 using HATU and HOAt, which
did produce the desired dipeptide 14, but in a disappointing 13%
yield.
With the protected dipeptides 11 and 14 in hand, we turned
our attention to the guanidinylation. Treatment of 11 with
lithium hydroxide in water–acetonitrile resulted in simultaneous
ester hydrolysis and Fmoc cleavage to give dipeptide 15 in
94% yield (Scheme 5). Guanidinylation of 15 using bis(tert-
butoxycarbonyl)triflylguanidine proceeded smoothly to afford the
dipeptide 16 in 90% yield.
Scheme 3 Reagents and conditions: i. TFA, CH2Cl2, 16 h; ii. Et3N,
CH2Cl2, BocGlyF, 16 h, 66% over 2 steps.
Scheme 5 Reagents and conditions: i. LiOH, MeCN–H2O, 16 h, 94%; ii.
Bis(tert-butoxycarbonyl)triflylguanidine, MeOH, 3 days, 90%.
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Table 1 Characterisation data for peptides
Repeating the hydrolysis step on dipeptide 14 surprisingly led
to removal of both Boc groups from the guanidino moiety, in
addition to methyl ester hydrolysis and Fmoc cleavage, yielding
the deprotected dipeptide 17 in 94% yield (Scheme 6). The use
of arginine with an unprotected side-chain is not uncommon
in peptide synthesis,24 so, rather than attempt to re-protect the
guanidino function, we simply went on to guanidinylate the amino
group of our isostere, leading to 18 in 52% yield.
Peptide
tr/min
m/z founda
m/z calc
GXaaAFVTIGK 19
RXaaIYDLIEL 20
GRAFVTIGK 21
RRIYDLIEL 22
25.83d
31.87d
11.42c
45.33b
961.2
1202.6
948.5
960.6
1202.7
948.6
1190.4
1190.7
a MALDI or electrospray, m/z for [M + H]+. b 70 : 30 Water–MeCN +
0.05% TFA. c 88 : 12 Water–MeCN + 0.05% TFA to 55 : 45 water–MeCN
+ 0.05% TFA over 30 min. d 99.95% Water + 0.05% TFA to 99.95% MeCN
+ 0.05% TFA over 60 minutes.
Scheme 6 Reagents and conditions: i. LiOH, MeCN–H2O, 16 h, 94%; ii.
Bis(tert-butoxycarbonyl)triflylguanidine, MeOH, 3 days, 52%.
Scheme 7 Reagents and conditions: i. DCC, DMAP, DMSO, 2 days; ii.
TFA, TES, phenol, H2O.
Peptide synthesis
a cell-surface class I MHC stabilisation assay; and a CD8 T-cell
cytotoxicity assay. The first provides a measure of the peptide–
MHC binding ability, while the second provides a measure of the
ability of an epitope-specific T-cell clone to recognise the peptide–
MHC complex displayed on the surface of a target cell.
Fragment coupling of the dipeptide 16 to the resin-bound hep-
tameric sequence AFVTIGK was attempted using HATU and
HOAt, but under these conditions the only product identified
following cleavage of the peptide from the support was the result
of resin-capping by the coupling reagent.25 Since epimerisation of
the quaternary centres in 16 and 18 is not an issue, more vigorous
coupling conditions may be used. In an attempt to increase the
reactivity of the dipeptide fragments, we used DCC in association
with the nucleophilic catalyst DMAP. The concentration was
important, and it was found that a dipeptide concentration of
0.2 M in DMSO, with a 48 hour coupling time at 60 ◦C, was
optimal. Under these conditions 16 coupled with resin-bound
AFVTIGK and 18 coupled with resin-bound IYDLIEL to afford
the desired nonameric peptides 19 and 20 in 42% and 47%
yield, respectively, after resin cleavage and HPLC purification
(Scheme 7). The two control sequences GRAFVTIGK 21 and
RRIYDLIEL 22 were synthesised by standard Fmoc peptide
synthesis protocols; the data for all four peptides are summarised
in Table 1.
Class I MHC stabilisation assays
The ability of the modified peptides and the native epitopes
to bind class I MHC molecules was assessed by a class I
MHC stabilisation assay,26 as described in the Experimental
section. Briefly, cells which are deficient in components of the
peptide-loading machinery, T2-B*2705, mostly present empty
HLA-B*2705 at the cell surface, which are unstable and are
rapidly degraded. Upon binding of an appropriate peptide, the
resultant complexes are stabilised and can be detected by a
fluorescently labelled conformation-specific monoclonal antibody,
W6/32. The fluorescence intensities of these labelled cells can then
be determined by flow cytometric analysis, giving a measure of the
ability of the peptide to stabilise the complex. This is expressed as
MHC stabilisation efficiency (MSE), the percentage increase of the
mean fluorescence above that of the negative control. All peptides
that resulted in a fluorescence intensity greater than the mean + 3
standard errors of the mean (SEM) of the fluorescence intensity
resulting from the T2-B*2705 cells in the absence of peptide at
Immunological testing
The modified peptides 19 and 20 were tested alongside the
native sequence peptides in two different immunological assays:
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26 ◦C (negative control) were considered to be positive binders. The
results of one representative assay of three are presented in Chart 1.
The peptides incorporating the isostere, 19 and 20, stabilised free
MHC molecules on the surface of T2-B*2705 cells (MSE 130.3
and 123.7 respectively). However, the modified peptides stabilised
free MHC molecules less well than the native epitopes 21 and
22 (MSE 178.2 and 171.4 respectively), although both gave MSE
values above the cut-off point for positive binders (DMSO + 3
SEM 109.8) and therefore do bind to HLA-B*2705.
Chart 2 51Cr release assays results at three effector–target cell ratios, 1 :
1, 2 : 1 and 5 : 1, represented as percentages.
specific cell lysis at an effector–target cell ratio of 5 : 1, indicating
that the complex formed between peptide 20 and HLA-B*2705 is
recognised by the T-cell clone, but the modified peptide elicits only
a weak response when compared to the native epitope.
Conclusion
We have successfully synthesised an a-methylated b,c-unsaturated
arginine analogue and incorporated it at position 2 of two peptide
epitopes presented by the class I MHC molecule HLA-B*2705.
Class I MHC stabilisation assays indicated that the modified
peptides have a lower affinity for HLA-B*2705 than the native
epitopes, contrary to our expectations, and chromium release
assays on one of the modified peptides confirm that it elicited
a weaker cytotoxic response from cognate epitope-specific CD8+
T-cells. It would appear that the incorporation of a double bond
and an a-methyl group into the arginine analogue is deleterious
to binding, although the presence of both features in the same
analogue prevent us from determining the effect of each on peptide
affinity for HLA-B*2705. It is possible that the effect of the a-
methyl substituent is to distort the backbone conformation of the
peptide, disrupting important binding interactions with the MHC
molecule.28 Further work will focus on peptidomimetics lacking
this feature.
Chart 1 MHC stabilisation on T2-B*2705 cells using peptides 19–22.
The dotted line (mean + 3 SEM) indicates the background fluorescence
intensity for T2-B*2705 cells incubated at 26 ◦C without peptide, which
was the cut-off for a positive result.
51Cr release assays
While cell-surface class I MHC stabilisation assays provide a
measure of peptide–MHC binding ability, they do not give any
information about the ability of the complex to be recognised
by the immune response. Chromium release cytotoxicity assays
can be used to determine the ability of peptide-loaded target cells
to be recognised by CD8+ T-cells.27 The target cells are loaded
with radioactive 51Cr before being incubated with the peptide.
On exposure to a T-cell clone specific for the particular peptide–
MHC complex, the target cells are lysed, releasing 51Cr which may
be measured using a scintillation detector; the amount of 51Cr
released provides a measure of the response of the T-cell clone
to the peptide–MHC complex. As we only had access to a T-cell
clone specific for the RRIYDLIEL–HLA-B*2705 complex, our
chromium release assays were limited to peptides 20 and 22.
51Chromium-loaded T2-B*2705 cells were used as target cells,
and these were sensitised in the presence of peptides 20 and 22 by
incubation for 1 hour at 37◦◦C in a 5% CO2 humidified atmosphere,
and then overnight at 26 C. Following incubation, the peptide-
sensitised cells were exposed to different concentrations of a T-cell
clone specific for the native RRIYDLIEL peptide epitope and the
resultant percentage specific cell lysis determined by measuring
the released radioactivity.
Experimental
General chemical procedures
THF and diethyl ether were distilled from sodium and benzophe-
none. Toluene, CH2Cl2, MeCN, triethylamine and diisopropy-
lethylamine were distilled from calcium hydride.
1H, 19F, and 13C NMR spectra (300, 282 and 75 MHz respec-
tively) were recorded on a Bruker AC-300 spectrometer. HSQC,
1
HMBC, COSY 90, H and 13C NMR spectra were recorded on
1
a Bruker DRX500 (500 MHz and 125 MHz for H and 13C) or
a Bruker AMX400 spectrometer (400 MHz and 100 MHz for
1H and 13C). 19F NMR spectra were referenced downfield from
fluorotrichloromethane. 1H and 13C NMR spectra were recorded
using deuterated solvent as the lock and were referenced downfield
from tetramethylsilane. 13C spectra NMR were recorded using the
PENDANT pulse sequence. J values are reported in Hz. The
Target cells sensitised with the native epitope 22 gave a high
percentage (44%, 23% and 18%) specific cell lysis for the effector–
target cell ratios 5 : 1, 2 : 1 and 1 : 1 respectively (Chart 2).
Cells sensitised with the modified peptide epitope 20 gave 16%
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t
multiplicities of the spectroscopic signals are represented in the
following manner; s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br s = broad singlet and env = overlapping signals.
Chemical ionisation (CI) and electron impact (EI) mass spectra
were recorded on a VG Zabspec mass spectrometer or a VG
Prospec mass spectrometer. Chemical ionisation (CI) methods
used ammonia as the carrier gas. Liquid secondary ion mass
spectrometry (LSIMS) was recorded using a VG Zabspec in-
strument. A Micromass LCT mass spectrometer was used for
both low-resolution electrospray time of flight (ES-TOF) mass
spectrometry (using a methanol mobile phase) and accurate mass
measurement (using a lock mass incorporated into the mobile
phase). Matrix assisted laser desorption ionisation time of flight
(MALDI-TOF) mass spectrometry was recorded using a Bruker
Biflex IV instrument using either sinapinic acid or (a-cyano-4-
hydroxy-trans-cinnamic acid–nitrocellulose [3 : 1]) as a thin layer
matrix.
(NHCO2 Bu), 173.9 (CO2Me); m/z (EI) 234 ([M + H]+, 100%),
202 ([M − CH2OH]+, 40).
Methyl (S)-2-(tert-butoxycarbonylamino)-3-oxo-2-methylpro-
panoate 5. To a solution of pyridinium chlorochromate (2.77 g,
12.85 mmol) in CH2Cl2 (50 mL) was added celite to form a slurry,
which was stirred vigorously for 5 minutes before being cooled
to 0 ◦C. A solution of alcohol 3 (2.0 g, 8.6 mmol) in CH2Cl2
(20 mL) was added, and the mixture was stirred overnight at room
temperature. Sodium metabisulfite (2 g) and Et2O (50 mL) were
added, and the mixture was stirred vigorously for 15 minutes.
The mixture was then filtered through a short pad of silica, dried
(MgSO4) and concentrated in vacuo to yield a pale yellow oil.
Purification by column chromatography (Rf = 0.42, petroleum
ether–Et2O 1 : 1) gave the aldehyde 5 as a colourless oil (1.07 g,
54%). [a]2D3 −11.4 (c 1.29 in CHCl3); mmax(CHCl3)/cm−1 3015, 2978,
1724, 1705, 1450, 1366, 1250, 1184, 1165, 1057; dH (300 MHz,
CDCl3) 1.39 (9H, s, C(CH3)3), 1.55 (3H, s, NHCCH3), 3.74 (3H,
s, CO2CH3), 5.62 (1H, s, NHCCH3), 9.51 (1H, s, CHO); dC
(75 MHz, CDCl3) 19.2 (NHCCH3), 28.1 (C(CH3)3), 52.3 (OCH3),
Thin layer chromatography was performed on precoated glass-
backed silica gel plates supplied by ICN Ltd (Silica gel 60 F254
,
thickness 0.25 mm). Column chromatography was performed on
silica gel 40–63 l 60A (Fluorochem Ltd).
t
66.6 (NHCCH3), 80.8 (C(CH3)3), 154.6 (NHCO2 Bu), 169.4
(CO2Me), 194.0 (CHO); m/z (ES) 286 ([M + Na + MeOH]+,
100%), 254 ([M + Na]+, 20), 230 ([M − H]+, 40).
All HPLC was performed using a Dionex Summit HPLC sys-
tem with Chromeleon software. Analytical and semi-preparative
HPLC were carried out using a Summit P580 quaternary low-
pressure gradient pump with built-in vacuum degasser. A P580P
high-pressure binary gradient pump with built-in vacuum degasser
was employed for preparative HPLC. A UVD 170s UV–VIS
multi channel detector was used to monitor all HPLC. Luna
10 l columns supplied by Phenomenex containing C18 as the
sorbent were used for all HPLC (250 × 4.6 mm, 250 × 10 mm and
250 × 21.2 mm columns were used for analytical, semi-preparative,
and preparative HPLC respectively). Unless otherwise stated, all
HPLC was performed using the following solvent mixtures: eluent
A (water–TFA (99.95 : 0.05)); and eluent B (MeCN–TFA (99.95 :
0.05)).
Methyl
(E,S)-2-(tert-butoxycarbonylamino)-5-oxo-2-methyl-
pent-3-enoate 6. To a solution of the aldehyde 5 (1.8 g,
7.8 mmol) in benzene (50 mL) was added (triphenylphos-
phoranylidene)acetaldehyde (3.56 g, 11.7 mmol) and the mixture
was stirred at room temperature for 4 days. Removal of the
solvent in vacuo and purification of the residue by column
chromatography (Rf = 0.35, petroleum ether–Et2O 1 : 1) gave the
unsaturated aldehyde 6 as a colourless oil (1.40 g, 70%). [a]2D3 −7.8
(c 0.36 in CHCl3); mmax(CHCl3)/cm−1 3018, 2974, 1713, 1706, 1518,
1425, 1216; dH (300 MHz, CDCl3) 1.41 (9H, s, C(CH3)3), 1.60
(3H, s, NHCCH3), 3.66 (3H, s, CO2CH3), 5.49 (1H, s, NHCCH3),
=
6.19 (1H, dd, J 15.8 and 7.7, CH CHCHO), 7.19 (1H, d, J 15.8,
CH CHCHO), 9.59 (1H, d, J 7.7, CHO); dC (75 MHz, CDCl3)
24.6 (NHCCH3), 28.4 (C(CH3)3), 53.4 (OCH3), 60.2 (NHCCH3),
80.9 (C(CH3)3), 131.1 (CH CHCHO), 154.6 (NHCO2 Bu), 156.3
=
Methyl (S)-2-(tert-butoxycarbonylamino)-3-hydroxy-2-methyl-
propanoate 3. ( )-a-Methylserine methyl ester hydrochloride16
(21.2 g, 0.16 mol) and (+)-CSA (37.0 g, 0.16 mol) were heated
at reflux in CH2Cl2 (200 mL) and acetone (23 mL) until a
homogeneous solution formed. The solution was allowed to cool
to room temperature and was left until crystallisation occurred.
The crystals were collected by filtration and the fractional crys-
tallisation procedure was repeated for a second time. The crystals
(23.4 g, 64 mmol) were dissolved in dry CH2Cl2 (100 mL) and Et3N
(8.9 mL, 64 mmol). (Boc)2O (13.97 g, 64 mmol) was added, and the
mixture was heated at reflux overnight. Following the addition of
water (50 mL), the organic layer was separated, dried (MgSO4) and
the solvent removed in vacuo to yield the enantiomerically enriched
alcohol 3 as a colourless oil (13.4 g, 68% overall yield, based on
t
=
=
(CH CHCHO), 172.3 (CO2Me), 193.6 (CHO); m/z (ES) 312
([M + Na + MeOH]+, 20%), 280 ([M + Na]+, 100).
Methyl (E,S)-2-(tert-butoxycarbonylamino)-5-hydroxy-2-methyl-
pent-3-enoate 7. To a solution of the unsaturated aldehyde 6
(3.19 g, 12.0 mmol) in MeOH (20 mL), CeCl3 (2.98 g, 14.0 mmol)
was added. The resultant mixture was stirred at room temperature
for 15 minutes, then sodium borohydride (0.47 g, 12.4 mmol) was
added and the reaction mixture was stirred overnight. Following
evaporation of the solvent in vacuo, water (10 mL) was added, and
the mixture was extracted with EtOAc (3 × 30 mL). The organic
phase was washed with brine, dried (MgSO4) and concentrated in
vacuo to yield a yellow oil. Purification by column chromatography
(Rf = 0.18, petroleum ether–Et2O 1 : 2) gave the allylic alcohol 7
as a colourless oil (1.6 g, 50%). [a]2D3 −10.7 (c 0.46 in CHCl3);
mmax(CHCl3)/cm−1 3364, 2978, 2950, 2869, 1712 (br), 1503, 1435,
1391, 1367, 1251, 1166, 1059; dH (300 MHz, CDCl3) 1.41 (9H, s,
C(CH3)3), 1.63 (3H, s, NHCCH3), 3.71 (3H, s, CO2CH3), 4.17 (2H,
d, J 4.6, CH2OH), 5.29 (1H, br s, NH), 5.72 (1H, dt, J 15.8 and 4.6,
25
recovery of a single enantiomer). [a] +1.5 (c 1.0 in CHCl3) (lit.18
589
18
[a] +1.9 (c 0.54 in CHCl3)), [a]25758 +3.1 (c 1.0 in CHCl3), [a]25546
589
+14.1 (c 1.0 in CHCl3), [a]24653 +23.6 (c 1.0 in CHCl3), [a]32655 +25.5
(c 1.0 in CHCl3); mmax(CHCl3)/cm−1 3348, 2978, 1750, 1724, 1450,
1366, 1250, 1184, 1165, 1057; dH (300 MHz, CDCl3) 1.37 (9H,
s, C(CH3)3), 1.41 (3H, s, NHCCH3), 3.60 (3H, s, OCH3), 3.61
(1H, d, J 9.9, CH2OH), 3.86 (1H, d, J 9.9, CH2OH), 5.42 (1H, s,
NH); dC (75 MHz, CDCl3) 19.6 (NHCCH3), 28.2 (C(CH3)3), 52.6
(OCH3), 60.9 (NHCCH3), 66.8 (CH2OH), 80.2 (C(CH3)3), 155.4
=
=
CH CHCH2), 5.89 (1H, d, J 15.8, CH CHCH2); dC (75 MHz,
CDCl3) 23.2 (NHCCH3), 28.3 (C(CH3)3), 52.8 (OCH3), 59.7
3774 | Org. Biomol. Chem., 2006, 4, 3769–3777
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49.37 minutes) as a white solid (0.26 g, 66%). [a]2D3 −25.9 (c 0.06
in CH3CN); mmax(film)/cm−1 3066, 2980, 1700 (br), 1520, 1450,
1254, 1167; dH (300 MHz, CD3CN) 1.40 (9H, s, C(CH3)3), 1.49
(3H, s, NHCCH3), 3.60–3.62 (5H, env, CH2NH, CO2CH3),
3.68–3.74 (2H, m, CH2NH), 4.20–4.26 (1H, m, CHCH2O), 4.33
(2H, d, J 7.0, CHCH2O), 4.79 (1H, br s, NH), 5.55 (1H, br s,
=
(NHCCH3), 62.8 (CH2OH), 80.8 (C(CH3)3), 130.0 (CH ), 131.1
t
=
(CH ), 154.4 (CO2 Bu), 173.4 (CO2Me); m/z (ES) 282 ([M +
Na]+, 100).
Methyl
(E,S)-2-(tert-butoxycarbonylamino)-5-(9H-fluoren-
9-ylmethoxycarbonylamino)-2-methylpent-3-enoate 8. To
a
solution of the allylic alcohol 7 (0.93 g, 3.59 mmol) in CH2Cl2
(20 mL), was added triethylamine (0.55 mL, 9.59 mmol) and
freshly distilled methanesulfonyl chloride (305 lL, 3.95 mmol),
and the mixture was stirred at room temperature overnight.
Following the addition of water (10 mL), the mixture was
extracted with CH2Cl2 (3 × 30 mL), washed with brine, dried
(MgSO4) and concentrated in vacuo to furnish the mesylate as a
colourless oil that was used immediately.
=
NH), 5.65 (1H, dt, J 15.4 and 4.8, CH CHCH2), 5.85 (1H,
=
d, J 15.4, CH CHCH2), 6.95 (1H, br s, NH), 7.35 (2H, dt, J
8.1 and 1.0, Ar–H), 7.42 (2H, dt, J 8.1 and 1.0, A–Hr), 7.65
(2H, d, J 8.1, A–Hr), 7.83 (2H, d, J 8.1, A–Hr); dC (75 MHz,
CD3CN) 22.9 (NHCCH3), 27.5 (C(CH3)3), 40.0 (CH2), 41.7
(CH2), 45.4 (CHCH2O), 50.4 (OCH3), 57.6 (NHCCH3), 64.4
(CHCH2O), (C(CH3)3) not detected, 119.9, 125.1, 127.1, 127.4,
=
127.7 (overlapping 4 Ar–CH, 2 CH ), 139.4 (Ar–C), 142.5 (Ar–
To a solution of the crude mesylate (1.44 g) in CH2Cl2 (20 mL)
was added ammonium hydroxide solution (28%, 10 mL), and the
mixture was stirred at room temperature for 4 h before the solvent
was removed in vacuo. The residue was dissolved in acetone–H2O
(10 : 1, 20 mL), NaHCO3 solution (1 M, 3.48 mL) and N-(9H-
fluoren-9-ylmethoxycarbonyloxy)succinimide (1.88 g, 5.6 mmol)
were added, and the mixture was stirred at room temperature
for 16 h. Following concentration in vacuo, the residue was
partitioned between H2O (50 mL) and CH2Cl2 (3 × 30 mL).
The combined organic extracts were washed with brine, dried
(MgSO4) and concentrated in vacuo. The residue was purified
by column chromatography (Rf = 0.35, hexane–EtOAc 2 : 1) to
yield an amorphous solid that was recrystallised from EtOAc–
hexane 2 : 1 to give the orthogonally protected amino ester 8 as
a white cryst◦alline solid (0.67 g, 25% overall yield for 3 steps).
Mp 110–112 C; [a]D23 −21.5 (c 0.02 in CHCl3); mmax(CHCl3)/cm−1
3017, 2977, 2950, 1742, 1712, 1513, 1449, 1367, 1215, 1164; dH
(300 MHz, CDCl3) 1.42 (9H, s, C(CH3)3), 1.57 (3H, s, NHCCH3),
3.73 (3H, s, CO2CH3), 3.80–3.88 (2H, m, CH2NH), 4.17–4.25 (1H,
m, CHCH2O), 4.40 (2H, d, J 7.0, CHCH2O), 4.73 (1H, br s, NH),
=
=
=
C), 154.5 (C O), 155.7 (C O), 167.9 (C O), 173.0 (CO2Me);
HRMS (ES) 560.2361 (C29H35N3O7Na requires 560.2373); m/z
(ES) 560 ([M + Na]+, 100).
N,Nꢀ,Nꢀꢀ-Tris(tert-butoxycarbonyl)arginine 13. To solution of
Na-tert-butoxycarbonylornithine (50 mg, 0.2 mmol) and iPr2EtN
(37 lL, 0.2 mmol) in MeOH (2 mL) was added bis(tert-
butoxycarbonyl)triflylguanidine (77 mg, 0.2 mmol) and the mix-
ture was stirred for 3 days. The solvent was removed in vacuo
and the residue purified by column chromatography (Rf = 0.42,
chloroform–methanol 19 : 1) to afford the protected amino acid
13 as a colourless oil (81 mg, 76%). [a]2D3 −79.2 (c 0.014 in
MeOH); mmax(CHCl3)/cm−1 3317, 2978, 1713, 1674, 1620, 1365,
1227, 1149; dH (300 MHz, CD3OD) 1.44 (9H, s, C(CH3)3), 1.46
(9H, s, C(CH3)3), 1.52 (9H, s, C(CH3)3), 1.63–1.73 (3H, env) and
1.78–1.90 (1H, m) (CHCH2CH2), 3.37 (2H, m, CH2NH), 4.09
(1H, m, COCH), 4.97 (3H, br s, 3 NH); dC (75 MHz, CD3OD)
28.2 (CH2), 29.7 (C(CH3)3), 30.1 (C(CH3)3), 30.2 (C(CH3)3), 31.5
(CH2), 42.8 (CH2), 56.2 (CH), 81.8 (C(CH3)3), 81.9 (C(CH3)3),
=
=
=
85.9 (C(CH3)3), 155.5 (C O), 158.9 (C O), 159.4 (C O), 165.8
+
=
=
4.91 (1H, br s, NH), 5.68 (1H, dt, J 15.8 and 5.5, CH CHCH2),
5.87 (1H, d, J 15.8, CH CHCH2), 7.30 (2H, t, J 7.7, Ar–H),
7.39 (2H, t, J 7.7, Ar–H), 7.58 (2H, d, J 7.7, Ar–H), 7.75 (2H,
d, J 7.7, Ar–H); dC (75 MHz, CDCl3) 24.4 (NHCCH3), 28.8
(C(CH3)3), 42.9 (CH2NH), 47.7 (CHCH2O), 52.9 (OCH3), 58.0
(NHCCH3), 67.3 (CHCH2O), 80.2 (C(CH3)3), 120.5, 125.5, 127.6,
(C N), 176.8 (CO2H); m/z (ES) 497 ([M + Na] , 100), 475 ([M +
H]+, 5%).
=
Methyl
(E,2S,2ꢀS)-2-{[N,Nꢀ,Nꢀꢀ-tris(tert-butoxycarbonyl)-
argininyl]amino}-5-(9H -fluoren-9-ylmethoxycarbonylamino)-2-
methylpent-3-enoate 14. Amino ester 8 (0.3 g, 0.63 mmol) was
deprotected by stirring in TFA–CH2Cl2 (1 : 2, 3 mL) overnight.
The solvent was removed in vacuo, iPr2EtN (0.22 mL, 1.25 mmol)
and THF (10 mL) were added, and the solution was stirred for 15
minutes. At the same time, the triprotected arginine 13 (61.39 mg,
0.39 mmol) was dissolved in THF (10 mL), and HATU (0.18 g,
0.69 mmol) and HOAt (94 mg, 0.69 mmol) were added. The
solution was stirred for 15 minutes before being added dropwise to
the solution of the deprotected amino ester, and the mixture was
stirred at room temperature overnight. Following filtration and
solvent removal in vacuo, the resulting residue was dissolved in
EtOAc. The solution was washed with 1 M citric acid, water and
brine, dried (MgSO4) and concentrated in vacuo. Purification by
HPLC (eluent A–B 100 : 0 to 0 : 100 over 60 minutes) yielded the
dipeptide 14 (tr 50.47 minutes) as a colourless oil (66 mg, 13%).
[a]D23 +26.7 (c 0.05 in CH3CN); mmax(neat)/cm−1 3284, 3083, 2966,
1710, 1632, 1538, 1203, 1139; dH (300 MHz, CD3CN) 1.39 (9H, s,
C(CH3)3), 1.46–1.54 (21H, env, 2 C(CH3)3, CCH3NH), 1.60–1.76
(4H, env, CHCH2CH2), 3.20–3.24 (1H, m, CHCH2CH2CHH),
3.37–3.45 (1H, m, CHCH2CH2CHH), 3.60 (3H, s, CO2CH3),
3.68–3.72 (2H, m, CH2NH), 4.08–4.12 (1H, m, BocNHCH),
=
=
128.2 (overlapping 4 Ar–CH, CH ), 132.7 (CH ), 141.8 (Ar–C),
=
=
144.4 (Ar–C), 152.6 (C O), 154.5 (C O), 171.5 (CO2Me); HRMS
(ES) 503.2148 (C27H32N2O6Na requires 503.2158); m/z (ES) 503
([M + Na]+, 100%), 447 ([M + Na–C4H8]+, 80).
Methyl (E,S)-2-{[(N-tert-butoxycarbonyl)glycyl]amino}-5-(9H-
fluoren-9-ylmethoxycarbonylamino)-2-methylpent-3-enoate
11.
Amino ester 8 (0.35 g, 0.73 mmol) was deprotected by stirring in
TFA–CH2Cl2 (1 : 2, 3 mL) overnight. Excess TFA was removed
in vacuo, the residue dissolved in CH2Cl2 (10 mL), and Et3N
(0.21 mL, 1.48 mmol) was added. The reaction mixture was
stirred for 10 minutes at room temperature before the addition
of N-(tert-butoxycarbonyl)glycyl fluoride (0.14 g, 0.82 mmol)
in CH2Cl2 (2 mL) over 60 seconds. The reaction was stirred at
room temperature for 16 h, then water (10 mL) was added, the
organic phase was separated and the aqueous phase extracted
with CH2Cl2 (3 × 10 mL). The combined organic extracts were
washed twice with 5% HCl, 10% NaHCO3, water, dried (MgSO4),
and concentrated in vacuo. Purification by HPLC (eluent A–B
100 : 0 to 0 : 100 over 60 minutes) yielded the dipeptide 11 (tr
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4.20–4.26 (1H, m, CHCH2O), 4.31 (2H, m, CHCH2O), 5.60–5.90
CD3OD) 1.53 (27H, br s, 3 × C(CH3)3), 1.60 (3H, s, NHCCH3),
=
=
=
(4H, env, CH CHCH2, CH CHCH2 and 2 NH), 7.20–7.28
(1H, m, NH), 7.34 (2H, t, J 7.4, Ar–H), 7.42 (2H, t, J 7.4, Ar–H),
7.64 (2H, d, J 7.4, Ar–H), 7.83 (2H, d, J 7.4, Ar–H), 9.30–9.35
(1H, m, NH), 9.58–9.67 (1H, m, NH); dC (75 MHz, CD3CN;
some doubling of signals evident due to restricted rotation) 23.0,
24.3, 24.8, 25.2, 25.4, 25.9, 29.1, 29.6, 41.3, 42.1, 42.4, 47.6, 52.6,
54.0, 59.8, 66.7, 82.5, 85.1, 85.5, 118.4, 123.5, 125.2, 125.9, 126.1,
126.7, 139.6, 142.9, 151.9, 153.0, 153.1, 155.1, 159.7, 160.2, 171.9,
173.2; HRMS (ES) 859.4197 (C43H60N6O11Na requires 859.4218);
m/z (ES) 859 ([M + Na]+, 50%), 557 (100).
3.92 (2H, d, J 5.9, CH CHCH2), 4.12 (2H, s, NHCH2CO)
=
5.73 (1H, dt, J 15.8, 5.9 CCH CHCH2), 6.17 (1H, d, J 15.8,
=
CCH CHCH2); dC (125 MHz, CD3OD) 24.3 (NHCCH3), 28.1
(3 C(CH3)3), 43.6 (CH2), 44.7 (CH2), 61.1 (NHCCH3), 85.9 and
=
=
86.0 (3 C(CH3)3), 124.4 (CH ), 134.4 (CH ), 153.5, 155.5, 156.0,
=
=
162.1, 167.5 (4 C O, C N), 175.3 (CO2H); m/z (ES) 566 ([M +
Na]+, 10%), 544 ([M + H]+, 40), 444 ([M + H–C5H8O2]+, 100).
(E,2S,2ꢀS)-2-{[(Na -tert-Butoxycarbonyl)argininyl]amino}-5-
{[(tert-butoxycarbonyl)amino-(tert-butoxycarbonylimino)methyl]-
amino}-2-methylpent-3-enoic acid 18. To a solution of the
dipeptide 17 (49 mg, 0.12 mmol) and iPr2EtN (21 lL,
0.12 mmol) in MeOH (2 mL), was added bis(tert-
butoxycarbonyl)triflylguanidine (44 mg, 0.12 mmol), and the
mixture was stirred at room temperature for 3 days. Evaporation
of the solvent in vacuo and purification by HPLC (eluent A–B
100 : 0 to 0 : 100 over 60 minutes) yielded the dipeptide 18
(tr 54.54 minutes) as a white solid (40 mg, 52%). [a]2D3 +33.9
(c 0.02 in CH3OH); mmax(KBr)/cm−1 3273, 3082, 2964, 1670,
1628, 1531, 1203, 1137; dH (500 MHz, CD3OD) 1.45 (9H, s,
C(CH3)3), 1.53 (9H, s, C(CH3)3), 1.54 (9H, s, C(CH3)3), 1.59 (3H,
s, CCH3NH), 1.67–1.82 (4H, env, CHCH2CH2), 3.18–3.22 (1H,
m, CHCH2CH2CHH), 3.35–3.39 (1H, m, CHCH2CH2CHH),
(E,S)-5-Amino-2-{[(N -tert-butoxycarbonyl)glycyl]amino}-2-
methylpent-3-enoic acid 15. The dipeptide 11 (115 mg,
0.71 mmol) was dissolved in MeCN (5 mL). LiOH (25.6 mg,
3.55 mmol) in H2O (0.5 mL) was added, and the mixture was
stirred at room temperature overnight. Removal of the solvent in
vacuo and purification by HPLC (eluent A–B 100 : 0 to 0 : 100 over
60 minutes) yielded the dipeptide 15 (tr 18.30 minutes) as a white
solid (61 mg, 94%). [a]2D3 −54.8 (c 0.02 in D2O); mmax(KBr)/cm−1
3281, 3088, 2968, 1675, 1633, 1536, 1457, 1203, 1139; dH (300 MHz,
D2O) 1.35 (9H, s, C(CH3)3), 1.48 (3H, s, NHCCH3), 3.55 (2H, d,
J 6.6, CH2NH2), 3.72 (2H, s, NHCH2CO), 5.59–5.71 (1H, m,
=
=
CH CHCH2), 6.23 (1H, d, J 15.8, CH CHCH2); dC (75 MHz,
D2O) 22.4, 26.5, 41.5, 58.3, 60.1, 85.3, 120.4, 134.7, 150.2, 165.4,
175.2; HRMS (ES) 324.1537 (C13H23N3O5Na requires 324.1535);
m/z (ES) 346 ([M − H + 2Na]+, 40%), 324 ([M + Na]+, 100), 302
([M + H]+, 20).
=
3.81–3.85 (1H, m, CHCHH), 4.01–4.08 (2H, env, BocNHCH,
=
=
CHCHH), 5.65–5.73 (1H, m, CH CHCH2), 6.07–6.16 (1H, m,
=
CH CHCH2); dC (125 MHz, CD3OD) 24.2 (CCH3NH), 24.8
(CHCH2CH2), 28.1 (C(CH3)3), 28.7 (C(CH3)3), 28.9 (C(CH3)3),
=
30.0 (CHCH2), 42.2 (CHCH2CH2CH2), 43.6 ( CHCH2), 55.8
(BocNHCH), 60.8 (NHCCH3), 81.0 (C(CH3)3), 85.8 (C(CH3)3),
(E,2S,2ꢀS )-5-Amino-2-{[(Na-tert-butoxycarbonyl)argininyl]-
amino}-2-methylpent-3-enoic acid 17. The dipeptide 14 (66 mg,
0.08 mmol) was dissolved in MeCN (3 mL). LiOH (9.4 mg,
0.39 mmol) in water (1 mL) was added, and the mixture was stirred
at room temperature overnight. Removal of the solvent in vacuo
and purification by HPLC (eluent A–B 100 : 0 to 0 : 100 over 60
minutes) yielded the dipeptide 17 (tr 26.24 minutes) as a white solid
(30 mg, 94%). [a]D23 −15.7 (c 0.02 in CH3CN); mmax(KBr)/cm−1 3276,
3093, 2964, 1673, 1650, 1531, 1205, 1137; dH (300 MHz, CD3OD)
1.45 (9H, s, C(CH3)3), 1.59 (3H, s, CCH3NH), 1.60–1.74 (4H,
env, CHCH2CH2), 3.17–3.21 (1H, m, CHCH2CH2CHH), 3.31–
3.37 (1H, m, CHCH2CH2CHH), 3.55 (2H, d, J 6.3, CH2NH2),
=
=
85.9 (C(CH3)3), 124.1 (CH CHCH2), 134.7 (CH CHCH2),
=
=
153.5, 153.6, 155.5, 157.9, 162.9, 163.2 (4 C O, 2 C N), 174.2
(CO2H); HRMS (ES) 643.3771 (C28H51N8O9 requires 643.3779);
m/z (ES) 687 ([M − H + 2Na]+, 60%), 665 ([M + Na]+, 10), 643
([M + H]+, 100), 543 ([M + H–C5H8O2]+, 70).
Manual peptide coupling and cleavage procedure
To a solution of the dipeptide (30 lmol) in DMSO (150 lL) at
room temperature were added DCC (7 mg, 33 lmol) and DMAP
(0.4 mg, 0.3 lmol). The resultant solution was stirred for 15
minutes before being added to the resin-bound heptameric peptide
(30 lmol; resin loadings were typically around 0.4 mmol g−1). The
reaction was heated at 60 ◦C for 2 days under an argon atmosphere.
Upon completion of the reaction the resin was transferred to a
cleavage vessel consisting of a quick fit glass tube with glass sinter
and three-way tap.17 The resin was washed with CH2Cl2 and then
treated with reagent B (TFA–phenol–water–triisopropylsilane [88 :
5 : 5 : 2, 10 mL]) at room temperature for 2 hours. The cleavage
cocktail was removed by filtration and the resin washed with TFA
(2 × 5 mL). The combined filtrate was evaporated to dryness and
the residue was triturated with Et2O (5 mL). The resultant solid
was dissolved in water (5 mL) and lyophilised to afford the fully
deprotected crude peptide as a white solid.
=
4.03–4.10 (1H, m, BocNHCH), 5.65–5.80 (1H, m, CH CHCH2),
=
6.29 (1H, d, J 15.8, CH CHCH2); dC (100 MHz, CD3OD; some
doubling of signals evident due to restricted rotation) 23.5, 24.2,
25.6, 26.3, 28.1, 28.7, 29.8, 30.2, 41.8, 42.0, 42.2, 53.7, 55.7, 60.7,
81.0, 85.8, 122.5, 137.9, 153.6, 158.0, 161.7, 162.0, 174.1, 175.1;
HRMS (ES) 401.2459 (C17H33N6O5 requires 401.2434); m/z (ES)
401 ([M + H]+, 100%), 301 ([M + H–C5H8O2]+, 55).
(E,2S) - 2{[(N - tert - Butoxycarbonyl)glycyl]amino} - 5 - {[(tert-
butoxycarbonyl)amino-(tert-butoxycarbonylimino)methyl]amino}-
2-methylpent-3-enoic acid 16. To a solution of the dipeptide 15
(40 mg, 0.13 mmol) and iPr2EtN (23 lL, 0.13 mmol) in MeOH
(2 mL) was added bis(tert-butoxycarbonyl)triflylguanidine
(47 mg, 0.13 mmol), and the mixture was stirred at room
temperature for 3 days. Removal of the solvent in vacuo and
purification by HPLC (eluent A–B 100 : 0 to 0 : 100 over 60
minutes) yielded the dipeptide 16 (tr 58.29 minutes) as a white
solid (64 mg, 90%). [a]D23 −14.8 (c 0.01 in MeOH); mmax(KBr)/cm−1
3434, 3057, 2924, 1722, 1601, 1451, 1273, 1117; dH (500 MHz,
Preparation of H–Gly–Xaa–Ala–Phe–Val–Thr–Ile–Gly–Lys–
OH 19. Following manual coupling and cleavage, purification
by HPLC (eluent A–B 100 : 0 to 0 : 100 over 60 minutes) gave
peptide 19 (tr 18.57 minutes) as a white solid (12 mg, 42%). m/z
3776 | Org. Biomol. Chem., 2006, 4, 3769–3777
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(MALDI) 983.2 ([M + Na]+, 100%), 961.2 ([M + H]+, 40) (calc.
960.6 for [M + H]+).
M. L. Salgaller, S. Southwood, P. F. Robbins, A. Sette, S. A. Rosenburg
and Y. Kawakami, J. Immunol., 1996, 157, 2539–2548; S. A. Rosenberg,
J. C. Yang, D. J. Schwartzentruber, P. Hwu, F. M. Marincola, S. L.
Topalian, N. P. Restifo, M. E. Dudley, S. L. Schwarz, P. J. Spiess, J. R.
Wunderlich, M. R. Parkhurst, Y. Kawakami, C. A. Seipp, J. H. Einhorn
and D. E. White, Nat. Med., 1998, 4, 321–327.
5 H. G. Rammensee, T. Friede and S. Stevanovic, Immunogenetics, 1995,
41, 178–228.
6 R. A. Sayle and E. J. Milner-White, Trends Biochem. Sci., 1995, 20,
374–376.
Preparation of H–Arg–Xaa–Ile–Tyr–Asp–Leu–Ile–Glu–Leu–
OH 20. Following manual coupling and cleavage, purification
by HPLC (eluent A–B 100 : 0 to 0 : 100 over 60 minutes) gave
peptide 20 (tr 31.87 minutes) as a white solid (17 mg, 47%). m/z
(MALDI) 1202.6 ([M + H]+, 100) (calc. 1202.7 for [M + H]+).
7 For alternative approaches to the design of high affinity ligands for
HLA-B*2705 see: D. Rognan, L. Scapozza, G. Folkers and A. Daser,
Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 753–757; G. A. Weiss, R. J.
Valentekovich, E. J. Collins, D. N. Garboczi, W. S. Lane, S. L. Schreiber
and D. C. Wiley, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 10945–10948;
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8 For a related approach to the design of high affinity ligands for the
class II MHC molecule HLA-DR1 see: A. B. Smith, A. B. Benowitz,
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10 T. S. Jardetzky, W. S. Lane, R. A. Robinson, D. R. Madden and D. C.
Wiley, Nature, 1991, 353, 326–329.
Cell surface stabilisation assays
T2-B*2705 cells (2 × 105) were incubated in serum-free AIM-
V medium in the presence of 100 lg mL−1 of the peptide for
14 to 16 h at 26 ◦C, after which the cells were incubated at
37 ◦C for 2 h prior to immunofluorescent staining. Cells were
washed free of unbound peptide with growth medium prior
to the addition of primary antibody. Anti-MHC allele-specific
monoclonal antibody, W6/32, was added to the T2-B*2705 cells
and incubated at 4 ◦C for 30 minutes. To detect binding of
the W6/32 monoclonal antibody, these cells were washed and
incubated with an anti-mouse fluorescein isothiocyanate labelled
antibody at 4 ◦C for 30 minutes. Finally, cells were washed and
resuspended in 500 lL of cold PBS supplemented with 1% FCS. A
sample of T2-B*2705 cells was incubated with AIM-V medium
alone (no peptide) at 26 ◦C for 14 to 16 h and served as a negative
control. The second negative control comprised a sample of T2-
B*2705 cells that had been cultured in growth medium without
11 J. M. Brooks, R. A. Colbert, J. P. Mear, A. M. Leese and A. B.
Rickinson, J. Immunol., 1998, 161, 5252–5259.
12 G. B. E. Stewart-Jones, K. di Gleria, S. Kollnberger, A. J. McMichael,
E. Y. Jones and P. Bowness, Eur. J. Immunol., 2005, 35, 341–351.
13 R. M. Williams, Synthesis of Optically Active a-Amino Acids, Pergamon
Press, Oxford, 1989.
◦
peptide at 37 C. Fluorescence intensities of the T2-B*2705 cells
were then measured using flow cytometry. The MHC stabilisation
efficiency (MSE) for each peptide was calculated as the percentage
increase of the mean fluorescence above that of the negative
controls.
14 R. M. Williams, Synthesis of Optically Active a-Amino Acids, Pergamon
Press, Oxford, 1989, pp. 22–28.
15 For two recent syntheses, and leading references, see: N. D. Smith and
M. Goodman, Org. Lett., 2003, 5, 1035–1037; J. W. Lane and R. L.
Halcomb, Org. Lett., 2003, 5, 4017–4020.
16 C. J. Easton, A. J. Ivory and C. A. Smith, J. Chem. Soc., Perkin Trans.
2, 1997, 503–507.
Cytotoxic assays (51Cr)
17 J. A. Dale, D. L. Dull and H. S. Mosher, J. Org. Chem., 1969, 34,
Target cells were sensitised with the synthetic peptides 20 and 22 at
a concentration of 5 lM during incubation with 0.7 mCi 51Cr for
90 minutes at 37 ◦C. Following incubation, these cells were washed
twice in growth medium and used as targets for cognate effector
cells at three effector ratios 1 : 1, 2 : 1 and 5 : 1 in a standard 5 hour
51Cr-release assay.27
2543–2549.
18 S. Hatakeyama, H. Matsumoto, H. Fukuyama, Y. Mukugi and H. Irie,
J. Org. Chem., 1997, 62, 2275–2279.
19 J. L. Luche, J. Am. Chem. Soc., 1978, 100, 2226–2227.
20 M. Goodman, C. Zapf and Y. Rew, Biopolymers, 2001, 60, 229–245.
21 L. A. Carpino, E.-S. M. E. Mansour and D. Sadat-Aalaee, J. Org.
Chem., 1991, 56, 2611–2614.
22 W. C. Chan and P. D. White, in Fmoc Solid Phase Peptide Synthesis, ed.
W. C. Chan and P. D. White, Oxford University Press, Oxford, 2000,
pp. 59–60.
Acknowledgements
23 K. Feichtinger, C. Zapf, H. L. Sings and M. Goodman, J. Org. Chem.,
We thank the Engineering and Physical Sciences Research Council
and the University of Birmingham for a studentship to M. A. J.,
and Professor Alan Rickinson for helpful discussions.
1998, 63, 3804–3805.
24 J. H. Jones, The Chemical Synthesis of Peptides, Oxford University
Press, Oxford, 1991, p. 77.
25 W. C. Chan and P. D. White, in Fmoc Solid Phase Peptide Synthesis, ed.
W. C. Chan and P. D. White, Oxford University Press, Oxford, 2000,
pp. 57–58.
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