Nonapeptide analogues containing (R)-3-hydroxybutanoate and β- homoalanine oligomers: Synthesis and binding affinity to a class I major histocompatibility complex protein

Article abstract of DOI:10.1021/jm981123l

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to t

Full text of DOI:10.1021/jm981123l

2318
J . Med. Chem. 1999, 42, 2318-2331
Articles
Non a p ep tid e An a logu es Con ta in in g (R)-3-Hyd r oxybu ta n oa te a n d â-Hom oa la n in e
Oligom er s: Syn th esis a n d Bin d in g Affin ity to a Cla ss I Ma jor
Histocom p a tibility Com p lex P r otein
Sorana Poenaru,‡,§ J ose´ R. Lamas,† Gerd Folkers,| J ose´ A. Lo´pez de Castro,† Dieter Seebach,*,‡ and
Didier Rognan*,|
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology, Universita¨tstrasse 16, CH-8092 Zu¨rich, Switzerland,
Centro de Biologia Molecular ”Severo Ochoa”, Facultad de Ciencias, Universidad Autonoma de Madrid,
E-28049 Madrid, Spain, and Department of Pharmacy, Swiss Federal Institute of Technology, Winterthurerstrasse 190,
CH-8057 Zu¨rich, Switzerland
Received December 4, 1998
Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical
modifications of the central part of the bound peptide should not alter binding affinity to the
MHC restriction protein but could perturb the T-cell response to the parent epitope. In our
effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of
(R)-3-hydroxybutanoate and(or) â-homoalanine have been substituted for the central part of a
HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the
B*2705 allele was determined by an in vitro stabilization assay. Four out of the six designed
analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing
the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer,
bound to B*2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly
interact with the protein active site, there are configurations which are not accepted by the
MHC binding groove, probably because of improper orientation of some lateral substituents in
the bound state and different conformational behavior in the free state. However we
demonstrate that â-amino acids can be incorporated in the sequence of viral T-cell epitopes
without impairing MHC binding. The presented structure-activity relationships open the door
to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists
aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.
In tr od u ction
independent recognition in which both terminal ends
of the peptide backbone are tightly bonded to conserved
residues of the MHC binding groove. Allele specificity
is ensured by the interaction of anchoring side chains,3,4
usually at positions P2, P3 (Pn standing for position n),
and the C-terminus with polymorphic pockets5 of the
host MHC protein. The central part of the bound
peptides (from positions 4 to 8) generally zigzags6 or
bulges7 out of the binding groove and thus allows
variation in the length of the bound peptides (from 8 to
11 amino acids). Systematic peptide mutation8 and
X-ray structure of MHC-peptide-TCR ternary com-
plexes9-12 show that this central part whose conforma-
tion is not complementary to that of the MHC protein
is the major contact area for Râ TCRs that trigger the
T-cell response to the foreign peptide.
Class I major histocompatibility complex (MHC)-
encoded proteins play a key role in the intracellular
immune surveillance by selectively binding to intracel-
lular peptide antigens and presenting them at the cell
surface to T-cell receptors (TCRs) of cytotoxic T-lym-
phocytes (CTL).1 Due to the genetically encoded dis-
crepancy between the limited number of class I alleles
(about six) expressed by each individual and the infinite
number of potential antigenic peptides (usually non-
amers), class I MHC molecules must bind diverse sets
of foreign peptides with a broad specificity but a high
affinity. Numerous structural data on class I MHC-
peptide complexes are nowadays available at the three-
dimensional level2 and provide an explanation for that
paradigm. The 27 reported X-ray structures (for nine
different class I MHC molecules) illustrate a peptide-
The tight association observed between MHC expres-
sion and susceptibility or resistance to autoimmune
disorders led us to consider class I MHC proteins as
particularly interesting targets for the selective immu-
notherapy of autoimmune diseases. At least two ways
of shunting the T-cell response to autoantigens using
small-molecular-weight molecules have been proposed.
The first one involving MHC blockade by a high-affinity
* To whom correspondence should be addressed. D. Seebach: fax,
+41.1.632 11 44; e-mail, seebach@org.chem.ethz.ch. D. Rognan: fax,
+41.1.635 68 84; e-mail, didier@pharma.ethz.ch.
‡ Laboratory for Organic Chemistry, SFIT.
† Universidad Autonoma de Madrid.
| Department of Pharmacy, SFIT.
§ Present address: Department of Chemistry, University of Cali-
fornia, Berkeley, CA 94720-1460.
10.1021/jm981123l CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/08/1999

Nonapeptide Analogue Binding Affinity to MHC Protein
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2319
Sch em e 1a
competitor13 is unlikely as MHC-bound peptides at the
cell surface are almost impossible to displace.14 The only
way to overcome this drawback would be to supply the
peptide competitor in liposomes15 or as lipopeptides16
into the endoplasmic reticulum where assembly of the
class I MHC-peptide complexes takes place. The second
inhibiting pathway relying on TCR antagonism17 sug-
gests that the presentation of the epitope to autoreactive
T-cells would be antagonized by a modified peptide
analogue. This approach is much more promising since
only a few TCRs at the surface of CTLs need to be
targeted,18 whereas MHC blockade requires saturation
of all MHC binding sites at the cell surface.19 Two
prerequisites are however necessary for designing TCR
antagonists: (i) a good affinity to the MHC-restriction
protein, (ii) a fast dissociation of the corresponding
MHC-ligand complex to the TCR.20,21 The few TCR
antagonists known to date are all peptide analogues for
which one TCR-anchoring amino acid has been mu-
tated.22 Unfortunately, the poor stability and pharma-
cokinetic properties inherent to their peptidic nature
preclude their general use as immunosuppressors. Thus,
there is a need for designing high-affinity nonpeptide
ligands for class I MHC proteins. Rather few variations
around the canonical nonapeptide structure have been
described up to date.23 Peptides bearing unnatural L-
or D-R-amino acids at MHC-anchoring positions,24-29
reduced peptide bond pseudopeptides,30 retroinverso
analogues,31 poly-N-acylated amines,32 or incorporation
of a â-homoglycine residue at the peptide N-terminus33
have been reported. An alternative strategy we initiated
3 years ago is to replace the central TCR-binding amino
acids by various nonpeptidic spacers: oligomers of
aminoalkanoates,24,34 phenanthridine derivatives,35 or
poly(ethylene glycol) loops.36 All these chemical modi-
fications led to ligands that could associate with class I
MHC proteins but always with a slight decrease in
binding affinity when compared to that of the parent
peptides.
a
(a) DCC, DMAP, CH2Cl2; (b) HCl/ether (satd); (c) H2, Pd/C,
MeOH; (d) Boc-Ala-OH, HOBt, EDC, Et3N, CH2Cl2; (e) H2, Pd/C,
MeOH; (f) HCl•H-Lys(Z)-OBn, HOBt, EDC, Et3N, CH2Cl2; (g) HCl/
dioxane (satd); (h) HOBt, EDC, DIEA, CH2Cl2; (i) TFA/CH2Cl2,
1:1.
thermore, combining â-HAla and HB oligomers should
enhance the solubility of the resulting spacers in
chlorinated solvents42 and thus facilitate their synthetic
access. Most of the designed peptide analogues bind
indeed with a high affinity to a class I MHC protein
(HLA-B*2705 allele), whose expression is associated
with susceptibility to severe autoimmune diseases.43
We recently described the replacement of a natural
pentameric peptide sequence (from positions P4 to P8)
by (R)-3-hydroxybutanoate (R-HB) oligomers in HLA-
B27-binding nonapeptides37 while enhancing 5-fold the
binding affinity for the MHC restriction protein.34
However, the partial hydrolysis of oligo-HB ester bonds,
observed during the synthesis, suggests that these
analogues should have very poor in vivo pharmacoki-
netic properties because of their high sensitivity to
esterases and peptidases. Recent reports on the remark-
able enzymatic stability of â-peptides38,39 led us to
consider oligomers of â-amino acids as potential sur-
rogates for the TCR-binding residues of class I MHC-
binding peptides. Since â-homoalanine (â-HAla) is an
isostere of HB, binding to HLA-B27 should thus be
retained in light of our previous results on poly(ester
peptides) (PEPs).34 However, the low solubility of pro-
tected â-HAla oligomers in any solvent40 could be a
drawback to the synthesis and biological evaluation of
these compounds. To increase the solubility in water of
compounds containing four â-HAla units,41 a positively
charged peptide epitope from the HIV-1 gp120 protein
(G314RAFVTIGK322, one-letter amino acid code), known
to bind well to HLA-B*2705,34 was chosen as template
for the reported chemical modifications (Table 1). Fur-
Resu lts a n d Discu ssion
Ch em ist r y. Synthesis of the derivative 11 was
achieved using a fragment-type coupling strategy
(Scheme 1). Boc-protected â-homoalanine38,44 and benzyl
3-hydroxybutanoate (2)45 were coupled using DCC and
DMAP as activating reagents46 to give 3. Deprotection
of the amino group under acidic conditions gave the
amino ester 4, whereas hydrogenolysis of the benzyl
ester gave acid 5. Coupling of 4 with the commercially
available Boc-protected alanine under traditional HOBt/
EDC peptide conditions47-49 gave the fully protected
derivative 6 whose benzyl ester group was cleaved by
1
H2 (Pd/C) to yield acid 7. H NMR measurements led
to assignment of all signals to the corresponding protons
of the R-amino acid, of the HB unit, and of the â-HAla
moiety.
To obtain the second fragment 9 with free amino and
protected carboxy group, the acid 5 was coupled with
H-Lys(Z)-OBn, using the HOBt/EDC strategy to give 8
(80%), treatment of which with a saturated HCl/dioxane
solution yielded the corresponding HCl salt 9. Com-
pound 10, consisting of six building blocks, was then

2320 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Poenaru et al.
Sch em e 2a
Sch em e 4a
a
(a) HCl•H-Lys(Z)-OBn, HOBt, EDC, Et3N; (b) HCl/dioxane
(satd); (c) 1, HOBt, EDC, DIEA, DMF; (d) HCl/dioxane (satd); (e)
Fmoc-Ala-OH, DCC, DMAP, CH2Cl2; (f) TFA/CH2Cl2, 1:1; (g)
HOBt, EDC, DIEA, CH2Cl2/DMF, 1:1; (h) Et2NH/DMF, 1:4.
Sch em e 3a
a
(a) Boc-Arg(NO2)-OH, HOBt, EDC, DIEA, DMF; (b) TFA, 10
min; (c) Boc-Gly-OH, HOBt, EDC, DIEA, DMF; (d) H2, Pd/BaSO4,
TFE/AcOH, 4:1; (e) TFA, 10 min.
application of the same fragment coupling strategy. The
fragment 15 was synthesized in a linear fashion,
coupling first Boc-â-HAla-OH (1) with H-Lys(Z)-OBn to
give dipeptide 12 (85%) which was deprotected to give
the HCl salt 13 (HCl in dioxane). The amino functional-
ity of 13, set free in situ by the base present in the
reaction mixture, was coupled with 1 to give the fully
protected compound 14 (74% from 12). The Boc group
of 14 was cleaved to yield 15. The fragment 17 was
obtained by ester formation between the hydroxy dimer
1637,51 and Fmoc-protected alanine (using DCC, DMAP),
and cleavage of the tert-butyl ester group (50% TFA)
gave the desired compound 18. It should be noted that
by using a small amount of DMAP (0.05 equiv), no
racemization was observed upon coupling.37 The amino
functionality, generated by in situ deprotonation of the
HCl salt 15, was coupled with the acid group of 18
(HOBt/EDC) to give the fully protected compound 19
in 92% yield. The Fmoc protecting group was then
cleaved (20% diethylamine in DMF52) to give the amino
ester 20. It is noteworthy that the backbone of com-
pound 19 varies from that of 10 only by the respective
positions of HB and â-HAla in the sequence. However
their respective solubilities in organic solvents are very
different. Compound 10 is highly soluble in chlorinated
a
(a) HCl•H-Lys(Z)-OBn, HOBt, EDC, DIEA, DMF; (b) TFA, 10
min; (c) Boc-Ala-OH, HOBt, EDC, DIEA, DMF; (d) TFA, 10 min.
obtained in 90% yield by coupling of 7 with 9.50
Subsequent cleavage of the Boc protecting group led to
the amino ester 11 which was used for the next coupling
with arginine, without further purification (Scheme 4).
The derivative 20, with HB and â-HAla incorporated
in different sequence (Scheme 2), was synthesized by

Nonapeptide Analogue Binding Affinity to MHC Protein
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2321
Ta ble 1. Binding and Analytical Properties of Ligands 27, 28, and 29a -d
a
compd
sequence
C50 (µmol)
HPLC (tR, min)b
MSc (M + 1)+
HIV gp120d
A68P1e
27
28
29a
29b
29c
29d
Gly-Arg-Ala-[P h e-Va l-Th r -Ile-Gly]-Lys
Glu-Val-Ala-Pro-Pro-Glu-Tyr-His-Arg
Gly-Arg-Ala-[(S-â-HAla -R-HB)2]-Lysg
Gly-Arg-Ala-[(R-HB)2-(S-â-HAla )2]-Lys
Gly-Arg-Ala-[(R-â-HAla )4]-Lys
Gly-Arg-Ala-[(S-â-HAla -R-â-HAla )2]-Lys
Gly-Arg-Ala-[(S-â-HAla )4]-Lys
Gly-Arg-Ala-[(R-â-HAla )2-(S-â-HAla )2]-Lys
2.8
nbf
2.8
18.7
19.5
16.4
15.4
15.3
15.4
773.3
774.1
771.9
772.6
771.9
772.0
17.0
2.8
4.8
6.0
30.0
a
Concentration of ligand at which HLA-B*2705 fluorescence (measured by FMC analysis with an anti-B27 monoclonal antibody) on
b
RMA-S cells was half the maximum obtained with that compound (see Experimental Section). HPLC purification using a gradient of A
(0.1% trifluoroacetic acid in water) and B (acetonitrile): 0-100% B, 60 min. c MALDI-TOF spectra, recorded on a Bruker Biflex instrument
(Bruker-Franzen Analytik, Bremen, Germany) in linear mode. HIV-1 glycoprotein 120 (314-322). e Self-peptide eluted from the HLA-
d
A68 allotype.8 f No detectable binding at 10-4 M. â-HAla, â-homoalanine; HB, 3-hydroxybutanoate.
g
solvents such as CH2Cl2, whereas 19 is poorly soluble
even in DMF.
The configurational isomers (diastereoisomers) 21a-d
containing four â-HAla units (Scheme 3) have been
previously prepared, starting from (S)- and (R)-Boc-Ala-
OH and using a fragment coupling strategy.40 These
oligomers are difficult to handle because of their low
solubility in any solvent tested so far. For example, the
1H NMR spectra of protected â-HAla tetramers can only
be measured using dimethyl-d6 sulfoxide as solvent and
show rather broad signals. During the synthesis, larger
amounts of DMF were necessary to dissolve the â-HAla
oligomers (c ) 0.05 M), as compared to those required
in R-peptide synthesis, and reaction times were conse-
quently longer. Therefore, the yields of the reactions are
not specified, since no purification is possible before the
last deprotection step, due to the poor solubility of this
class of compounds.
The acids 21 were coupled with H-Lys(Z)-OBn using
HOBt/EDC, to provide the fully protected derivatives,
the Boc protecting groups of which were cleaved (con-
centrated TFA) to give the TFA salts 22 which were
precipitated with ether and dried in high vacuum before
the next reaction step: deprotonation and reaction with
Boc-Ala-OH to yield, after another deprotection step, the
corresponding TFA salts 23.
During the cleavage of the Boc group, the Z protecting
group of the lysine side chain was partially cleaved,
giving rise to a byproduct (e5%) which has been
detected by mass spectrometry. Such debenzylations
F igu r e 1. Dynamic properties of complexes of B*2705 with
two modified peptides (29b, 29d ) and the reference HIV-1
gp120 (314-322) peptide. (A) Intermolecular hydrogen-bond
have been previously reported by Merrifield et al.53
However, considering the much harsher conditions used
by the Merrifield group, the observed loss of the Z group,
frequencies, recorded for the whole 500-ps trajectory of HLA-
in our case, was surprising. Due to solubility problems,
it was impossible to purify the intermediates at this
stage. Thus, we have carried the byproduct all along
the following synthetic steps, with the consequence that
some additional impurities were formed. After the last
deprotection step, preparative HPLC purification still
gave the desired pure compounds (27-29). The amino
functionalities of the derivatives 11, 20, and 23a -d
(Scheme 4) were allowed to react with Boc-Arg(NO2)-
OH,54 using the same coupling procedure as for the
other coupling steps. Again, the Boc groups of the fully
protected derivatives were cleaved with TFA to yield
compounds 24, 25, and 26a -d which, in turn, were
coupled with Boc-Gly-OH to give the fully protected
nonapeptides analogues. The NO2 and Z protecting
groups, as well as the benzyl ester group, were then
simultaneously removed by hydrogenation, using Pd/
B27-ligand complexes over 1000 conformations. Frequencies
between 25% and 50% and higher than 50% are displayed as
white and gray columns, respectively. (B) Buried surface areas
of HLA-B27-bound ligands 29b (white columns) and 29d (gray
columns) calculated on energy-minimized time-averaged con-
formations. Surface areas were calculated using the MS
program74 with a 1.4-Å radius probe.
BaSO4 as catalyst.56 Subsequent treatment with TFA
led to cleavage of the Boc groups to give, after HPLC
purification, the desired nonapeptide analogues 27, 28,
and 29a -d which were used for binding assays.
Bin d in g Affin ity to HLA-B*2705. The binding
affinities of the modified peptides clearly show that the
chirality of the spacer is important for recognition of
the B*2705 protein. Compounds in which the chiral
building block linked with the C-terminus (PC) has (R)-
configuration (27, 29a -b) were all potent ligands with

2322 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Poenaru et al.

Nonapeptide Analogue Binding Affinity to MHC Protein
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2323
binding affinities similar to that of the parent HIV-1
gp120 peptide (Table 1). This observation is in agree-
ment with our previous report on PEPs for which a
penultimate (R)-HB unit was proposed to interact with
the MHC binding groove.34 Analogues bearing a moiety
of (S)-configuration next to PC were less active, espe-
cially compounds 28 and 29d sharing the same sequence
of (R,R,S,S)-configuration of the spacing oligomers
(Table 1). However, an (S)-chiral spacer attached to PC
does not necessarily prevent binding (see compound 29c,
Table 1). Furthermore, it seems that certain configura-
tions of the four spacing monomers are detrimental to
binding. Thus, the two weakest binders (28, 29d ) share
the same (R,R,S,S)-configuration at positions 4 to 7.
Replacing ester by amide groups in the spacer (cf. 27
with 29b and 28 with 29d ) did not affect binding for
both high-affinity and low-affinity ligands. This result
is in agreement with the available X-ray structure56 of
a B*2705-nonapeptide complex, showing weak contri-
butions of peptide bonds, located between the P4 and
P8 positions, to the binding of a nonapeptide to HLA-
B27.
tween the two nonnatural complexes could be correlated
with the H-bond-donating strength of the N-terminus,
well-known to significantly contribute to the binding
free energy of nonapeptides to class I MHC proteins.58
The buried surface areas of each monomer of the
protein-bound ligand were also very similar with the
exception of two residues P5, and PC (Figure 1B). P5
corresponds to the second unit of the spacer (R-âHAla
in both cases). Depending on its environment in the
sequence of the modified peptide, it is more or less
deeply buried in the HLA-B27 binding groove. With
compound 29b, the P5 position is significantly deeper
inside the groove than with compound 29d (compare
Figure 2A,B). However, this feature is unlikely to induce
nearly a 10-fold difference in the binding affinity of the
corresponding ligands. The C-terminal amino acid (Lys)
also shows a better fit in the case of the high-affinity
ligand (Figure 1B). As the C-terminal residue is an
important anchor to B*2705,56 this structural difference
should also contribute to the improved binding of 29b
versus 29d .
However, the computed properties of the two ana-
logues bound to their target protein can only explain a
part of the experimentally determined difference of
binding affinities. The modeling study presented here
takes into account only enthalpic contributions to the
binding of each ligand to HLA-B*2705. As desolvation
energies and rotational/translational entropy losses
upon binding (assuming a conserved binding mode of
the two compounds) should be very similar due to the
structural analogy of all modified peptides listed in
Table 1, the 10-fold decreased binding of two analogues
(28, 29d ), having the same configuration, may be due
to different association rates and different conforma-
tional populations in the free state. This feature has
already been experimentally described for two related
PEPs,34 for which the length of the polyester spacer
varies. Hydrophobic â-peptides are known to have
conformations strongly depending on the chirality of
their residues and on the nature of their side chains.38,59
The (R,R,S,S)-configuration of four chiral monomers in
low-affinity ligands might lead to a conformational space
arrangement in the free state that is different from that
of high-affinity compounds (27, 28, 29a -c). The higher
“strain energy” necessary to bring ligands 28 and 29d
from the free to the bound state may partially contribute
to the weaker binding of these two analogues. Unfor-
tunately, simulating the free ligands, although compu-
tationally easier, is very risky because they adopt no
stable secondary structure, as concluded from their CD
or NMR spectra.
Apart from binding potencies, it should be noticed
that HB-containing compounds 27 and 28 are probably
still sensitive to esterases, although stability studies on
these compounds have not been performed yet. We also
expect that the replacement of HB oligomers by â-amino
acids in analogues 29a -d enhances the resistance of
the modified peptides to enzymatic degradation.
Molecu la r Mod elin g of B*2705-Liga n d Com -
p lexes. To find a rational explanation for the weak
binding of compounds with (R,R,S,S)-configuration of
the chiral spacer, a 500-ps molecular dynamics (MD)
study of the binary complexes between B*2705 and
three ligands was undertaken. Compound 29b was
chosen as representative of the high-affinity peptides,
whereas 29d was selected as representative of weak
binding ligands. The parent HIV peptide was selected
as reference. The trajectory of the three solvated com-
plexes was stable after 350 ps, with rms deviations of
the protein backbone from the starting X-ray coordi-
nates of ca. 1.5 Å (data not shown). We previously used
atomic fluctuations of the bound ligands, as a criterion,
for discriminating high-affinity from low-affinity pep-
tides.24,33,57 In the present case, they were very similar
for ligands 29b and 29d . Thus, subtle differences must
cause the very different binding affinities of the two
modified peptides. In fact, recording the frequency of
the MHC-ligand hydrogen bonds allows to distinguish
the two modified peptides. High-affinity ligands (HIV
gp120, 29b) present many more hydrogen bonds to the
HLA-B27 binding groove than the weak binding com-
pound 29d (Figure 1A). Strong H-bonds with a fre-
quency higher than 50% were remarkably identical in
both cases, but medium H-bonds (with frequencies
between 25% and 50%) are significantly in favor of 29b.
A very similar pattern has already been observed for a
set of four natural peptides binding to two closely
related HLA-B27 alleles.33 The major differences be-
Con clu sion
Replacing the central amino acids of class I MHC-
binding peptides by (R)-3-hydroxybutanoate and(or)
â-homoalanine oligomers leads to still high-affinity
ligands. Up to now, â-amino acids have hardly been used
in medicinal chemistry. Some natural â-amino acids
(taurine, â-aminobutyric acid, â-aminoisobutyric acid)
F igu r e 2. Three-dimensional structure of HLA-B*2705 in complex with 29b (A) and 29d (B). Peptide positions are labeled at
the CR atom from 1 (P1) to 8 (P8). The backbone trace of the MHC antigen-binding domain (R1, R2) of the B*2705 protein is
represented as bands (R helices), arrows (â strands), and tubes (coil). Altered peptides are displayed by a ball-and-stick model. A
white arrow indicates the position of the second â-ΗAla unit in both chiral spacers. The figure has been prepared using the
MOLSCRIPT75 and RASTER3D76 programs.

2324 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Poenaru et al.
DIEA, 3 equiv). HOBt (1.25 equiv), the acid (1 equiv), and EDC
(1.25 equiv) were then successively added. The reaction
mixture was allowed to warm to room temperature and then
stirred for 18 h. The mixture was diluted with CH2Cl2 and
washed with 1 N HCl, saturated NaHCO3 solution, and brine.
The organic layer was dried over anhydrous MgSO4, filtrated,
and concentrated. The resulting residue was purified on silica
gel to afford the pure product.
have been reported as agonists of the inhibitory glycine
receptor.60 Substituted â-amino acids have also been
described as fibrinogen receptor GIIb/IIIa antagonists,61
â-lactamase inhibitors,62 µ-opioid receptor agonists,63 or
enkephalin-degrading enzyme inhibitors.64 Further-
more, various â-amino acids are found in natural
antibiotics, fungicides, and antineoplastic compounds.65
However, to the best of our knowledge, this is the very
first report of biologically active molecules containing
â-amino acid oligomers. The present study demonstrates
that â-amino acids are valuable tools, indeed, for
designing peptidomimetics of bioactive peptides. By
contrast to most R-amino acid surrogates, the H-bonding
properties of backbone atoms, the backbone direction,
or the side chain directionality might be similar in
natural and â-peptides, at the condition that the â-pep-
tide can adopt the biologically active conformation of its
natural R-peptide analogue. Thus, if all side chains are
not mandatory for biological activity, â-amino acids and,
more generally, â-peptides undoubtedly represent new
promising tools in medicinal chemistry. In the special
case of class I MHC ligands, one might imagine to use
â-amino acids for replacing MHC anchors and(or) TCR
contact residues. Such altered peptides may thus lead
to peptide-based vaccines and TCR antagonists, which
would be stable to all common peptidases tested so far,
including pronase, 20S proteasome, and proteinase K.
Gen er a l P r oced u r e B: Am in o Acid Cou p lin g. The free
amine or the appropriate salt (1 equiv) was dissolved in DMF
(0.15 M) under argon and cooled to 0 °C. The reaction mixture
was treated with DIEA (3 equiv). HOBt (1.25 equiv), the acid
(1 equiv), and EDC (1.25 equiv) were then successively added.
The product was precipitated by the addition of a saturated
NaHCO3 solution. The precipitate was washed several times
with saturated NaHCO3, 1 M KHSO4 solutions and H2O and
dried 24 h under high vacuum to give the crude product which
was utilized in the next step without further purification.
Gen er a l P r oced u r e C: Boc Clea va ge Usin g a HCl
Solu tion . Under argon and at 0 °C, the Boc-protected com-
pound was dissolved in a saturated HCl/(EtO2 or dioxane)
solution. The mixture was stirred for 15 min to 1 h and then
evaporated. The resulting HCl salt was precipitated in ether,
dried under high vacuum, and used for the next step without
further purification.
Gen er a l P r oced u r e D: Boc Clea va ge Usin g a TF A
Solu tion . Under argon and at 0 °C, the Boc-protected com-
pound was dissolved in a TFA/CH2Cl2 (50-100%) solution. The
mixture was stirred for 10 min to 1 h and then evaporated.
The resulting TFA salt was precipitated in Et2O, dried under
high vacuum, and used for the next step without further
purification.
Exp er im en ta l Section
Gen er a l P r oced u r e E: F in a l Dep r otection . The fully
protected compound was dissolved in TFE/CH3COOH (3/1),
and a catalytic amount of 10% Pd/BaSO4 was added. The
apparatus was evacuated and flushed three times with H2, and
the mixture was stirred under an atmosphere of H2 for ca. 15
h. The mixture was then filtrated though Celite, concentrated,
and precipitated from Et2O. The resulting yellow-white CH3-
COOH salt was then treated with concentrated TFA. After 15
min, the crude product was precipitated from Et2O and
purified by HPLC.
Abbr evia tion s: (R)-â-homoalanine (R-â-HAla), (S)-â-ho-
moalanine (S-â-HAla), dicyclohexylcarbodiimide (DCC), diiso-
propylethylamine (DIEA), 4-(dimethylamino)pyridine (DMAP),
dimethylformamide (DMF), N-(3-(dimethylamino)propyl)-N′-
ethylcarbodiimide hydrochloride (EDC), 1-hydroxy-1H-benzo-
triazole (HOBt), trifluoroacetic acid (TFA), trifluoroethanol
(TFE), (R)-3-hydroxybutanoate (R-HB).
Ch em istr y. Dichloromethane (CH2Cl2) was dried over 4-Å
molecular sieves. Solvents for chromatography and workup
procedures were distilled from Sikkon (anhydrous CaSO4,
Fluka). Triethylamine (Et3N) was distilled from CaH2 and
stored over KOH. Amino acid derivatives were purchased from
Bachem or Senn. All other reagents were used as received from
Fluka.
Boc-S-â-HAla -R-HB-OBn (3). To a solution of the (R)-3-
hydroxybutanoic benzyl ester (2)45 (1.90 g, 9.8 mmol) in CH2-
Cl2 (30 mL) was added a solution of the acid 138 (2.00 g, 9.8
mmol) in CH2Cl2 (30 mL) under argon, cooled to -5 °C. DCC
(2.12 g, 10.3 mmol) and DMAP (0.09 g, 0.49 mmol) were added.
The resulting mixture was allowed to warm to room temper-
ature and then stirred for 24 h. The mixture was diluted with
Et2O and washed with 1 N HCl, saturated NaHCO3 solution,
and brine. The organic layer was dried over anhydrous MgSO4,
filtrated, and concentrated. The residue was purified on silica
gel (20% Et2O/pentane) and gave compound 3 (3.25 g, 88%)
as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 7.37-7.33
(m, 5H ar), 5.36-5.26 (m, 1H, CHO), 5.14 (AB, J ) 12.1, 1H,
OCH2Ph), 5.09 (AB, J ) 12.1, 1H, OCH2Ph), 5.00-4.90 (m,
1H, NH), 4.10-3.96 (m, 1H, CHN), 2.66 (dd, ABX, J ) 7.5,
15.6, 1H, CH2CHO), 2.54 (dd, ABX, J ) 5.3, 15.6, 1H, CH2-
CHO), 2.43 (dd, ABX, J ) 5.3, 14.9, 1H, CH2CHN), 2.37 (dd,
ABX, J ) 5.9, 14.9, 1H, CH2CHN), 1.43 (s, 9H, tBu), 1.29 (d,
J ) 6.2, 3H, Me of HB), 1.17 (d, J ) 6.5, 3H, Me of â-HAla).
13C NMR (75 MHz, CDCl3): δ 171.00, 170.42, 155.36, 135.93,
128.84, 128.60, 67.58, 66.65, 43.60, 40.77, 28.46, 20.31, 19.96.
FAB-MS: m/z 759 {26, (2M + 1)+}, 308 {64, (M + 1)+}, 280
(100).
1H (300-MHz) and 13C (75-MHz) NMR spectra were recorded
on a Varian Gemini 300 spectrometer and are reported in ppm
on the δ scale from TMS. Coupling constants are reported in
hertz (Hz). FAB-MS spectra were obtained with a Hitachi
Perkin-Elmer RHU-6M using a 3-nitrobenzyl alcohol (3-
NOBA) matrix. Elemental analyses were performed by the
Microanalytical Laboratory of the Laboratorium fu¨r Orga-
nische Chemie, ETH-Zu¨rich (only analyses above 0.4% were
given).
Chromatography generally refers to flash silica gel 60 (Fluka
40-63 mm) and TLC (Merck Kieselgel 60 F254 plates), detec-
tion with UV and ninhydrin. HPLC analyses were carried out
on a C18 analytical column on a Knauer HPLC system (pump
type 64, EuroChrom 2000 integration package, degaser, UV
detector (variable-wavelength monitor)) using a linear gradient
of (A) 0.1% CF3COOH in H2O and (B) MeCN at a flow rate of
1 mL/min with UV detection at 220 nm. HPLC purification
was carried out on a C8 preparative column on a Knauer
HPLC system (pump type 64, programmer 50, UV detector
(variable-wavelength monitor)) using a gradient of (A) 0.1%
CF3COOH in H2O and (B) MeCN at a flow rate of 4 mL/min
with UV detection at 214 nm. Retention times (tR) are given
in min.
HCl•H-S-â-HAla -R-HB-OBn (4). According to general
procedure C, compound 3 (227 mg, 0.6 mmol) was treated with
a saturated HCl/Et2O solution (6 mL). The resulting HCl salt
4 was obtained as a white precipitate and used in the next
coupling step without further purification.
Gen er a l P r oced u r e A: Am in o Acid Cou p lin g. The free
amine or the appropriate salt (1 equiv) was dissolved in CH2-
Cl2 or 50% CH2Cl2/DMF (0.1 M) under argon and cooled to 0
°C. The reaction mixture was treated with a base (Et3N or
Boc-S-â-HAla -R-HB-OH (5). The benzyl-protected com-
pound 3 (900 mg, 2.9 mmol) was dissolved in MeOH (20 mL);
catalytic amounts of 10% Pd/C (90 mg) and acetic acid (0.1

Nonapeptide Analogue Binding Affinity to MHC Protein
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2325
mL) were added. The apparatus was evacuated and flushed
three times with H2, and the mixture was stirred under an
atmosphere of H2 for ca. 8 h. Subsequent filtration though
Celite and concentration under reduced pressure yielded the
acid 5 (558 mg, 84%) as a colorless oil which was identified by
NMR and used for the next coupling step without purification.
) 6.2, 3H, Me), 1.17 (d, J ) 6.8, 3H, Me), 1.15 (d, J ) 6.5, 3H,
Me). 13C NMR (75 MHz, CDCl3): δ 172.49, 170.79, 170.08,
169.58, 156.98, 136.84, 135.64, 128.86, 128.78, 128.73, 128.54,
128.37, 128.29, 68.61, 68.27, 67.19, 66.74, 52.39, 42.47, 42.21,
41.24, 40.40, 31.42, 29.40, 28.41, 22.36, 20.06, 19.85, 19.71,
18.81. FAB-MS: m/z 906 {6, (M + Na)+}, 884 {32, (M + 1)+},
784 (100). Anal. (C45H65N5O13•H2O) C, H, N.
TF A•H-Ala -(S-â-HAla -R-HB)2-Lys(Z)-OBn (11). Accord-
ing to general procedure D, compound 10 (654 mg, 0.74 mmol)
was treated with a CH2Cl2/TFA (1:1) solution (6 mL). After
30 min, the reaction was completed and the solvent was
evaporated. The TFA resulting salt 11 was obtained in almost
quantitative yield as a white precipitate (from Et2O) and used
in the next coupling step without further purification.
Boc-Ala -S-â-HAla -R-HB-OBn (6). According to general
procedure A, to a solution in CH2Cl2 (26 mL) of HCl salt 4 (1
equiv, 2.61 mmol) was added Et3N (1.45 mL, 10.4 mmol). HOBt
(440 mg, 3.3 mmol), Boc-Ala-OH (4.94 mg, 2.61 mmol), and
then EDC (623 mg, 3.3 mmol) were successively added to the
reaction. The residue was purified by recrystallization (Et2O/
pentane, 2/5) to give compound 6 (994 mg, 85%) as a white
1
solid. H NMR (300 MHz, CDCl3): δ 7.35-7.326 (m, 5H ar),
6.70 (br d, J ) 6.8, 1H, NH), 5.33-5.27 (m, 1H, CHO), 5.12 9
(s, 2H, OCH2Ph), 5.12-5.06 (m, 1H, NH), 4.36-4.22 (m, 1H,
CHN), 4.18-4.06 (m, 1H, CHN), 2.74-2.66 (m, 1H, CH2CHO),
2.58 (dd, ABX, J ) 5.0, 15.57, 1H, CH2CHO), 2.42 (d, J ) 5.3,
2H, CH2CHN), 1.44 (s, 9H, tBu), 1.34-1.29 (m, 6H, 2 Me), 1.17
(d, J ) 6.8, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.12,
170.87, 135.80, 128.86, 128.67, 128.62, 67.87, 66.78, 42.02,
40.69, 40.33, 28.38, 19.98, 19.66, 18.64. FAB-MS: m/ z 901 {11,
(2M + 1)+}, 451 {100, (M + 1)+9}, 351 (48).
Boc-Ala -S-â-HAla -R-HB-OH (7). The benzyl-protected
compound 6 (675 mg, 1.5 mmol) was dissolved in MeOH (10
mL); catalytic amounts of 10% Pd/C (70 mg) and acetic acid
(0.1 mL) were added. The apparatus was evacuated and
flushed three times with H2, and the mixture was stirred under
an atmosphere of H2 for ca. 8 h. Subsequent filtration though
Celite and concentration under reduced pressure yielded the
acid 7 in almost quantitative yield as a colorless oil which was
used for the next coupling step without purification.
Boc-S-â-HAla -Lys(Z)-OBn (12). According to general pro-
cedure A, to a solution in DMF (50 mL) of the HCl salt of Lys-
(Z)-OBn (2.00 g, 4.9 mmol) was added Et3N (2.04 mL, 14.7
mmol). HOBt (0.83 g, 6.1 mmol), the acid Boc-â-HAla-OH (1)
(1.00 g, 4.9 mmol), and then EDC (1.17 g, 6.1 mmol) were
successively added to the reaction. The residue was purified
by recrystallization (ethyl acetate/hexane, 20/1) to give com-
1
pound 12 (2.30 g, 85%) as a white solid. H NMR (300 MHz,
CDCl3): δ 7.37-7.26 (m, 10H ar), 6.48-6.36 (m, 1H, NH),
5.22-5.10 (m, 3H, OCH2Ph, NH), 5.09 (s, 2H, OCH2Ph), 4.95-
4.87 (m, 1H, NH), 4.62-4.56 (m, 1H, CHN), 4.00-3.90 (m, 1H,
CHN), 3.17-3.10 (m, 2H, CH2NHZ), 2.46-2.32 (m, 2H, CH2-
CHN), 1.90-1.60 (m, 2H, CH2), 1.50-1.25 (m, 4H, CH2), 1.42
(s, 9H, tBu), 1.16 (d, J ) 6.8, 3H, Me). 13C NMR (75 MHz,
CDCl3): δ 172.44, 156.90, 136.85, 135.56, 128.89, 128.78,
128.65, 128.37, 67.29, 66.74, 52.01, 44.18, 42.52, 40.41, 31.61,
29.30, 28.46, 22.15, 20.60. FAB-MS: m/z 556 {28, (M + 1)+},
456 (100).
HCl•H-S-â-HAla -Lys(Z)-OBn (13). According to general
procedure C, compound 12 (1.00 g, 1.8 mmol) was treated with
a saturated HCl/dioxane solution (20 mL). After 30 min, the
reaction was completed and the solvent was evaporated. The
resulting HCl salt 13 was obtained in almost quantitative yield
as a white precipitate (from Et2O) and used in the next
coupling step without further purification.
Boc-S-â-HAla -R-HB-Lys(Z)-OBn (8). According to general
procedure A, to a solution in CH2Cl2 (15 mL) of HCl‚H-Lys-
(Z)-OBn (784 mg, 1.9 mmol) was added Et3N (1.08 mL, 7.7
mmol). HOBt (326 mg, 2.4 mmol), compound 5 (558 mg, 1.9
mmol), and then EDC (460 mg, 2.4 mmol) were successively
added to the reaction. The residue was purified on silica gel
(50% Et2O/pentane) to give compound 8 (964 mg, 78%) as a
white solid. 1H NMR (300 MHz, CDCl3): δ 7.38-7.29 (m, 10H
ar), 6.96-6.90 (m, 1H, NH), 5.27-5.17 (m, 1H, CHO), 5.22-
5.09 (m, 2H, OCH2Ph), 5.08 (s, 2H, OCH2Ph), 4.97-4.94 (m,
1H, NH), 4.90-4.86 (m, 1H, NH), 4.66-4.58 (m, 1H, CHN),
4.16-4.05 (m, 1H, CHN), 3.16-3.09 (m, 2H, CH2NHZ), 2.54-
2.27 (m, 4H, CH2CHN, CH2CHO), 1.90-1.60 (m, 4H, 2 CH2),
1.50-1.28 (m, 2H, CH2), 1.40 (s, 9H, tBu), 1.30 (d, J ) 6.2,
3H, Me), 1.13 (d, J ) 6.8, 3H, Me). 13C NMR (75 MHz,
CDCl3): δ 172.57, 170.84, 169.74, 156.83,155.65, 136.87,
135.69, 128.86, 128.76, 128.71, 128.55, 111.19, 79.61, 68.19,
67.16, 66.74, 52.19, 43.84, 42.30, 41.96, 40.61, 31.71, 29.46,
28.43, 22.42, 20.90, 19.46. FAB-MS: m/z 642 {12, (M + 1)+},
542 (100).
HCl•H-S-â-HAla -R-HB-Lys(Z)-OBn (9). According to gen-
eral procedure C, compound 8 (712 mg, 1.1 mmol) was treated
with a saturated HCl/dioxane solution (10 mL). After 15 min,
the reaction was completed and the solvent was evaporated.
The resulting HCl salt 9 was obtained in almost quantitative
yield as a white precipitate and used in the next coupling step
without further purification.
Boc-Ala -(S-â-HAla -R-HB)2-Lys(Z)-OBn (10). According to
general procedure A, to a solution in CH2Cl2 (15 mL) of the
HCl salt 9 (1 equiv, 1.5 mmol) was added DIEA (1.0 mL, 6.0
mmol). HOBt (253 mg, 1.8 mmol), compound 7 (1 equiv, 1.5
mmol), and then EDC (358 mg, 1.8 mmol) were successively
added to the reaction. The residue was purified on silica gel
(ethyl acetate/hexane, 9/1) to give compound 10 (884 mg, 90%)
as a white solid foam. 1H NMR (300 MHz, CDCl3): δ 7.36-
7.30 (m, 10H ar), 7.10-7.00 (m, 3H, NH), 5.34 (d, J ) 7.5, 1H,
NH), 5.30-5.10 (m, 3H, CHO, NH), 5.21-5.09 (m, 2H, OCH2-
Ph), 5.07 (s, 2H, OCH2Ph), 4.60-4.53 (m, 1H, CHN), 4.44-
4.31 (m, 2H, CHN), 4.17-4.08 (m, 1H, CHN), 3.16-3.08 (m,
2H, CH2NHZ), 2.55-2.29 (m, 8H, CH2CHN, CH2CHO), 1.88-
1.60 (m, 2H, CH2), 1.58-1.22 (m, 4H, CH2), 1.42 (s, 9H, tBu),
1.31 (d, J ) 6.8, 3H, Me), 1.29 (d, J ) 6.2, 3H, Me), 1.25 (d, J
Boc-(S-â-H Ala )2-Lys(Z)-OBn (14). According to general
procedure A, to a solution in DMF (5 mL) of the HCl salt 13
(1 equiv, 1.8 mmol) was added DIEA (0.92 mL, 5.4 mmol).
HOBt (304 mg, 2.2 mmol), the acid Boc-â-HAla-OH (1) (365
mg, 1.8 mmol), and then EDC (430 mg, 2.2 mmol) were
successively added to the reaction. The residue was purified
by recrystallization (CH3Cl/hexane, 20/1) to give compound 14
(850 mg, 74%) as a white solid. 1H NMR (300 MHz, CDCl3):
δ 7.38-7.26 (m, 10H ar), 6.68-6.65 (m, 2H, NH), 5.23-5.09
(m, 4H, OCH2Ph, NH), 5.09 (s, 2H, OCH2Ph), 4.58-4.50 (m,
1H, CHN), 4.30-4.20 (m, 1H, CHN), 4.00-3.90 (m, 1H, CHN),
3.18-3.10 (m, 2H, CH2NHZ), 2.42-2.22 (m, 4H, CH2CHN),
1.90-1.62 (m, 2H, CH2), 1.52-1.25 (m, 4H, CH2), 1.42 (s, 9H,
tBu), 1.19 (d, J ) 6.5, 3H, Me), 1.15 (d, J ) 6.8, 3H, Me). 13C
NMR (75 MHz, CDCl3): δ 172.44, 155.78, 128.88, 128.78,
128.62, 128.37, 67.30, 66.74, 52.27, 43.10, 40.29, 31.29, 28.56,
28.48, 22.21, 20.24. FAB-MS: m/z 663 {22, (M + Na)+}, 641
{38, (M + 1)+}, 541 (100).
HCl•H-(S-â-HAla )2-Lys(Z)-OBn (15). According to general
procedure C, compound 15 (712 mg, 1.1 mmol) was treated
with a saturated HCl/dioxane solution (10 mL). After 30 min,
the reaction was completed and the solvent was evaporated.
The HCl resulting salt 15 was obtained in almost quantitative
yield as a white precipitate (from Et2O) and used in the next
coupling step without further purification.
F m oc-Ala -(R-HB)2-OtBu (17). To a solution of the hydroxy
derivative 1651 (1 equiv, 4.4 mmol) in CH2Cl2 (40 mL) was
added Fmoc-Ala-OH (1.45 g, 4.4 mmol) under argon, and the
mixture was cooled to - 5 °C. DCC (0.95 g, 4.62 mmol) and
DMAP (0.04 g, 0.22 mmol) were added, and the resulting
mixture was allow to warm to room temperature and then
stirred for 24 h. The mixture was diluted with Et2O and
washed with 1 N HCl, saturated NaHCO3 solution, and brine.
The organic layer was dried over anhydrous MgSO4, filtrated,
and concentrated. The residue was purified on silica gel (Et2O/

2326 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Poenaru et al.
pentane, 2/3) to give compound 17 (700 mg, 30%) as a white
foam. 1H NMR (300 MHz, CDCl3): δ 7.77-7.75 (m, 2H ar),
7.61-7.59 (m, 2H ar), 7.42-7.37 (m, 2H ar), 7.34-7.26 (m,
2H ar), 6.49 (d, J ) 7.2, NH), 5.38-5.22 (m, 2H, CHO), 4.44-
4.32 (m, 3H, CHN, CH2 of Fmoc), 4.25-4.20 (m, 1H, CH of
Fmoc), 2.69-2.37 (m, 4H, CH2CHO), 1.43 (s, 9H, tBu), 1.30
(d, J ) 6.2, 3H, Me), 1.29 (d, J ) 6.2, 3H, Me), 1.26 (d, J )
6.5, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.44, 169.69,
169.37, 155.91, 144.23, 144.12, 141.58, 127.94, 125.33, 120.20,
81.10, 68.68, 68.21, 67.09, 49.83, 47.27, 40.05, 40.88, 28.09,
19.80, 19.67, 18.67. FAB-MS: m/z 540 {6, (M + 1)+}, 484 (100).
F m oc-Ala -(R-HB)2-OH (18). According to general proce-
dure C, compound 17 (600 mg, 1.1 mmol) was treated with a
TFA/CH2Cl2 (1:1) solution (6 mL). After 30 min, the reaction
was completed and the solvent was evaporated. The acid 18
was obtained in almost quantitative yield as a yellow oil and
used in the next coupling step without further purification.
soluble in any flash chromatography or HPLC solvent. The
presence of the desired Boc-(S-â-HAla-R-â-HAla)2-Lys(Z)-OBn
(11 mg, 90% crude) was confirmed by 1H and 13C NMR and
MS spectra. Further treatment with TFA (0.55 mL), according
to the general procedure D, gave the TFA salt 22b, which was
used without further purification.
TF A•H-(S-â-HAla )4-Lys(Z)-OBn (22c). According to gen-
eral procedure B, to a solution in DMF (15 mL) of HCl•H-Lys-
(Z)-OBn (515 mg, 1.27 mmol) was added DIEA (0.65 mL, 3.81
mmol). HOBt (214 mg, 1.59 mmol), the acid Boc-(S-â-HAla)4-
OH (21c)40 (1 equiv, 1.27 mmol), and then EDC (304 mg, 1.59
mmol) were successively added to the reaction. The resulting
precipitate was dried under high vacuum and used without
further purification as it is not soluble in any flash chroma-
tography or HPLC solvent. The presence of the desired Boc-
(S-â-HAla)4-Lys(Z)-OBn (819.1 mg, 79% crude) was confirmed
1
by H and 13C NMR and MS spectra. Further treatment with
TFA (4 mL), according to the general procedure D, gave the
TFA salt 22c, which was used without further purification.
F m oc-Ala -(R-HB)2-(S-â-HAla )2-Lys(Z)-OBn (19). Accord-
ing to general procedure A, to a solution in CH2Cl2/DMF (1:1,
12 mL) of the HCl salt 15 (1 equiv, 1.10 mmol) was added
DIEA (0.75 mL, 4.40 mmol). HOBt (185 mg, 1.37 mmol), the
acid 18 (1 equiv, 1.10 mmol), and then EDC (262 mg, 1.37
mmol) were successively added to the reaction. After 15 h
reaction, some of the product precipitated in the reaction
mixture. The CH2Cl2 was then evaporated, and the resulting
oil was precipitated in a saturated NaHCO3 solution. The
precipitate was washed several times with saturated NaHCO3,
KHSO4 (1 N) solutions and finally with H2O and dried 15 h
under high vacuum. The presence of compound 19 was
confirmed by 1H and 13C NMR and MS spectra. Compound 19
was used without further purification. 1H NMR (300 MHz,
DMSO-d6): δ 8.28-8.22 (m, 1H, NH), 7.30-7.94 (m, 1H, NH),
7.80-7.74 (m, 1H, NH), 7.75-7.65 (m, 2H ar), 7.42-7.24 (m,
16H ar), 7.22-7.16 (m, 1H, NH), 5.23-5.02 (m, 3H, CHO, NH),
5.208 (s, 2H, OCH2Ph), 5.96 (s, 2H, OCH2Ph), 4.32-4.14 (m,
2H, CHN), 4.10-4.22 (m, 2H, CHN), 2.96-2.87 (m, 2H, CH2-
NHZ), 2.40-2.00 (m, 8H, CH2CHN, CH2CHO), 1.70-1.51 (m,
2H, CH32), 1.40-1.10 (m, 4H, 2 CH2), 1.23 (d, J ) 6.8, 3H,
Me), 1.13 (d, J ) 5.0, 3H, Me), 1.04 (d, J ) 5.9, 3H, Me), 1.03-
0.98 (m, 2 Me). 13C NMR (75 MHz, DMSO-d6): δ 172.28,
170.52, 169.11, 168.09, 167.99, 156.26, 155.99, 144.02, 140.90,
137.43, 136.14, 128.55, 128.47, 128.15, 127.94, 127.84, 127.77,
127.21, 125.38, 120.25, 68.34, 67.77, 65.83, 65.64, 65.12, 63.44,
51.88, 49.42, 46.58, 44.25, 42.18, 42.05, 41.82, 41.64, 30.38,
28.86, 23.18, 22.55, 19.82, 19.75, 19.51, 19.38, 19.15. FAB-
MS: m/z 1028 {23, (M + Na)+}, 1006 {41, (M + 1)+}, 809 (84),
713 (100).
H-Ala -(R-HB)2-(S-â-HAla )2-Lys(Z)-OBn (20). The Fmoc-
protected compound 19 (520 mg, 0.52 mmol) was dissolved in
DMF/Et2NH (9:1, 4 mL) under argon and cooled to 0 °C. The
mixture was stirred for 1-2 h, and concentration under
reduced pressure yielded the crude amine 20 which was
identified by NMR and used without further purification.
TF A•H-(R-â-HAla )4-Lys(Z)-OBn (22a ). According to gen-
eral procedure B, to a solution in DMF (11 mL) of HCl•H-Lys-
(Z)-OBn (442 mg, 1.09 mmol) was added DIEA (0.56 mL, 3.27
mmol). HOBt (184 mg, 1.36 mmol), the acid Boc-(â-HAla)4-
OH (21a )40 (1 equiv, 1.09 mmol), and then EDC (260 mg, 1.36
mmol) were successively added to the reaction. The residue
was dried under high vacuum and used without further
purification as it is not soluble in any flash chromatography
or HPLC solvent. The presence of the desired Boc-(R-â-HAla)4-
Lys(Z)-OBn (723 mg, 82% crude) was confirmed by 1H and 13C
NMR and MS spectra. Further treatment with TFA (3.6 mL),
according to the general procedure D, gave the TFA salt 22a ,
which was used without further purification.
TF A•H-(R-â-HAla )2-(S-â-HAla )2-Lys(Z)-OBn (22d ). Ac-
cording to general procedure B, to a solution in DMF (11 mL)
of HCl•H-Lys(Z)-OBn (442 mg, 1.09 mmol) was added DIEA
(0.56 mL, 3.27 mmol). HOBt (184 mg, 1.36 mmol), the acid
Boc-(R-â-HAla)2-(S-â-HAla)2-Lys(Z)-OH (21d )40 (1 equiv, 1.09
mmol), and then EDC (260 mg, 1.36 mmol) were successively
added to the reaction. The resulting precipitate was dried
under high vacuum and used without further purification as
it is not soluble in any flash chromatography or HPLC solvent.
The presence of the desired Boc-(R-â-HAla)2-(S-â-HAla)2-Lys-
(Z)-OBn (796 mg, 90% crude) was confirmed by 1H and 13C
NMR and MS spectra. Further treatment with TFA (3.9 mL),
according to the general procedure D, gave the TFA salt 22d ,
which was used without further purification.
TF A•H-Ala -(R-â-HAla )4-Lys(Z)-OBn (23a ). According to
general procedure B, to a solution in DMF (7 mL) of the TFA
salt 22a (1 equiv, 0.89 mmol) was added DIEA (0.61 mL, 3.56
mmol). HOBt (150 mg, 1.11 mmol), Boc-Ala-OH (202 mg, 1.06
mmol), and then EDC (212 mg, 1.1 mmol) were successively
added to the reaction. The resulting precipitate was dried
under high vacuum and used without further purification as
it is not soluble in any flash chromatography or HPLC solvent.
The presence of the desired Boc-Ala-(R-â-HAla)4-Lys(Z)-OBn
1
(680 mg, 87% crude) was confirmed by H and 13C NMR and
MS spectra. Further treatment with TFA (3.1 mL), according
to the general procedure D, gave the TFA salt 23a , which was
used without further purification.
TF A•H-Ala -(S-â-HAla -R-â-HAla )2-Lys(Z)-OBn (23b). Ac-
cording to general procedure B, to a solution in DMF (2 mL)
of the TFA salt 22b (1 equiv, 0.135 mmol) was added DIEA
(0.092 mL, 0.540 mmol). HOBt (23 mg, 0.169 mmol), Boc-Ala-
OH (31 mg, 0.162 mmol), and then EDC (32 mg, 0.169 mmol)
were successively added to the reaction. The resulting pre-
cipitate was dried under high vacuum and used without
further purification as it is not soluble in any flash chroma-
tography or HPLC solvent. The presence of the desired Boc-
Ala-(S-â-HAla-R-â-HAla)2-Lys(Z)-OBn (73 mg, 61% crude) was
confirmed by 1H and 13C NMR and MS spectra. Further
treatment with TFA (0.33 mL), according to the general
procedure D, gave the TFA salt 23b, which was used without
further purification.
TF A•H-Ala -(S-â-HAla )4-Lys(Z)-OBn (23c). According to
general procedure B, to a solution in DMF (10 mL) of the TFA
salt 22c (1 equiv, 1.00 mmol) was added DIEA (0.68 mL, 4.00
mmol). HOBt (169 mg, 1.25 mmol), Boc-Ala-OH (227 mg, 1.20
mmol), and then EDC (239 mg, 1.25 mmol) were successively
added to the reaction. The resulting precipitate was dried
under high vacuum and used without further purification as
it is not soluble in any flash chromatography or HPLC solvent.
The presence of the desired Boc-Ala-(S-â-HAla)4-Lys(Z)-OBn
TF A•H-(S-â-HAla -R-â-HAla )2-Lys(Z)-OBn (22b). Accord-
ing to general procedure B, to a solution in DMF (2 mL) of
HCl•H-Lys(Z)-OBn (62 mg, 0.15 mmol) was added DIEA (0.08
mL, 0.45 mmol). HOBt (26 mg, 0.19 mmol), the acid Boc-(S-
â-HAla-R-â-HAla)2-OH (21b)40 (1 equiv, 0.15 mmol), and then
EDC (36 mg, 0.19 mmol) were successively added to the
reaction. The resulting precipitate was dried under high
vacuum and used without further purification as it is not
1
(762 mg, 86% crude) was confirmed by H and 13C NMR and
MS spectra. Further treatment with TFA (3.5 mL), according
to the general procedure D, gave the TFA salt 23c, which was
used without further purification.
TF A•H-Ala -(R-â-HAla )2-(S-â-HAla )2-Lys(Z)-OBn (23d ).

Nonapeptide Analogue Binding Affinity to MHC Protein
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2327
According to general procedure B, to a solution in DMF (10
mL) of the TFA salt 22d (770 mg, 0.93 mmol) was added DIEA
(0.64 mL, 3.72 mmol). HOBt (157 mg, 1.16 mmol), Boc-Ala-
OH (211 mg, 1.12 mmol), and then EDC (215 mg, 1.16 mmol)
were successively added to the reaction. The resulting pre-
cipitate was dried under high vacuum and used without
further purification as it is not soluble in any flash chroma-
tography or HPLC solvent. The presence of the desired Boc-
Ala-(R-â-HAla)2-(S-â-HAla)2-Lys(Z)-OBn (490 mg, 60% crude)
tography or HPLC solvent. The presence of the desired Boc-
Arg(NO2)-(S-â-HAla-R-â-HAla)2-Lys(Z)-OBn (74 mg) was con-
firmed by FAB-MS. Further treatment with TFA (0.5 mL),
according to the general procedure D, gave the TFA salt 26b,
which was used without further purification.
TF A•H-Ar g(NO2)-Ala -(S-â-HAla )4-Lys(Z)-OBn (26c). Ac-
cording to general procedure B, to a solution in DMF (9 mL)
of the TFA salt 23c (1 equiv, 0.86 mmol) was added DIEA (0.59
mL, 3.44 mmol). HOBt (145 mg, 1.07 mmol), Boc-Arg(NO2)-
OH (329 mg, 1.03 mmol), and then EDC (205 mg, 1.07 mmol)
were successively added to the reaction. The resulting pre-
cipitate was dried under high vacuum and used without
further purification as it is not soluble in any flash chroma-
tography or HPLC solvent. The presence of the desired Boc-
Arg(NO2)-Ala-(S-â-HAla)4-Lys(Z)-OBn (860 mg, 82% crude)
1
was confirmed by H and 13C NMR and MS spectra. Further
treatment with TFA (2.2 mL), according to the general
procedure D, gave the TFA salt 23d , which was used without
further purification.
TFA•H-Ar g(NO2)-Ala-(S-â-HAla-R-HB)2-Lys(Z)-OBn (24).
According to general procedure A, to a solution in CH2Cl2/DMF
(4:3, 8 mL) of the TFA salt 11 (1 equiv, 0.74 mmol) was added
DIEA (0.38 mL, 2.22 mmol). HOBt (125 mg, 0.92 mmol), Boc-
Arg(NO2)-OH (259 mg, 0.81 mmol), and then EDC (176 mg,
0.92 mmol) were successively added to the reaction. The
resulting residue was purified on silica gel (CH2Cl2/MeOH, 9/1)
to give compound Boc-Arg(NO2)-Ala-(S-â-HAla-R-HB)2-Lys(Z)-
OBn (650 mg, 82%) as a fine white powder. 1H NMR (300 MHz,
CDCl3): δ 8.45-8.30 (m, 1H, NH), 7.80-7.60 (m, 3H, NH),
7.8-7.28 (m, 10H ar), 7.20-7.12 (m, 3H, NH), 5.70-5.60 (m,
1H, NH), 5.34-5.26 (m, 1H, NH), 5.26-5.06 (m, 4H, CHO,
OCH2Ph), 5.07 (s, 2H, OCH2Ph), 4.60-4.50 (m, 1H, CHN),
4.46-4.2 (m, 4H, CHN), 3.36-3.24 (m, 2H, CH2NHC), 3.16-
3.07 (m, 2H, CH2NHZ), 2.53-2.33 (m, 8H, CH2CHN, CH2-
CHO), 1.90-1.60 (m, 6H, CH2), 1.54-1.10 (m, 4H, CH2) 1.41
(s, 9H, tBu), 1.34 (d, J ) 6.8, 3H, Me), 1.28 (d, J ) 6.2, 3H,
Me), 1.24 (d, J ) 6.5, 3H, Me), 1.20-1.16 (m, 6H, Me). 13C
NMR (75 MHz, CDCl3): δ 172.61, 170.35, 170.27, 169.77,
157.09, 136.82, 135.61, 128.87, 128.80, 128.54, 128.37, 128.23,
80.46, 68.68, 68.40, 67.22, 65.96, 52.48, 49.54, 42.48, 42.16,
40.46, 31.36, 29.41, 28.39, 24.85, 22.41, 20.02, 19.88, 19.74,
19.66, 18.19. FAB-MS: m/z 1107 {60, (M + Na)+}, 1085 {100,
(M + 1)+}, 985 (22), 713 (7). Further treatment with TFA (5
mL), according to the general procedure D, gave the TFA salt
24, which was used without further purification.
TF A•H-Ar g(NO2)-Ala -(R-HB)2-(S-â-HAla )2-Lys(Z)-OBn
(25). According to general procedure B, to a solution in DMF
(6 mL) of the TFA salt 20 (1 equiv, 0.52 mmol) was added
DIEA (0.26 mL, 1.55 mmol). HOBt (87 mg, 0.65 mmol), Boc-
Arg(NO2)-OH (198 mg, 0.62 mmol), and then EDC (123 mg,
0.65 mmol) were successively added to the reaction. The
resulting precipitate was dried under high vacuum and used
without further purification as it is not soluble in any flash
chromatography or HPLC solvent. The presence of the desired
Boc-Arg(NO2)-Ala-(R-HB)2-(S-â-HAla)2-Lys(Z)-OBn (409 mg,
77% crude) was confirmed by 1H and 13C NMR and MS spectra.
Further treatment with TFA (1.4 mL), according to the general
procedure D, gave the TFA salt 25, which was used without
further purification.
1
was confirmed by H and 13C NMR and MS spectra. Further
treatment with TFA (3.1 mL), according to the general
procedure D, gave the TFA salt 26c, which was used without
further purification.
TF A•H -Ar g(NO2)-Ala -(R-â-H Ala )2-(S-â-H Ala )2-Lys(Z)-
OBn (26d ). According to general procedure B, to a solution
in DMF (9 mL) of the TFA salt 23d (1 equiv, 0.45 mmol) was
added DIEA (0.31 mL, 1.81 mmol). HOBt (76 mg, 0.56 mmol),
Boc-Arg(NO2)-OH (172 mg, 0.54 mmol), and then EDC (107
mg, 0.56 mmol) were successively added to the reaction. The
resulting precipitate was dried under high vacuum and used
without further purification as it is not soluble in any flash
chromatography or HPLC solvent. The presence of the desired
Boc-Arg(NO2)-Ala-(R-â-HAla)2-(S-â-HAla)2-Lys(Z)-OBn (460 mg,
84% crude) was confirmed by 1H and 13C NMR and MS spectra.
Further treatment with TFA (2.2 mL), according to the general
procedure D, gave the TFA salt 26d , which was used without
further purification.
H-Gly-Ar g-Ala -(S-â-HAla -R-HB)2-Lys-OH (27). According
to general procedure B, to a solution in DMF (6 mL) of the
TFA salt 24 (1 equiv, 0.50 mmol) was added DIEA (0.26 mL,
1.51 mmol). HOBt (85 mg, 0.63 mmol), Boc-Gly-OH (97 mg,
0.55 mmol), and then EDC (124 mg, 0.63 mmol) were succes-
sively added to the reaction. The resulting precipitate was
dried under high vacuum and used without further purification
as it is not soluble in any solvent to be purified. The presence
of the desired Boc-Gly-Arg(NO2)-Ala-(S-â-HAla-R-HB)2-Lys-
(Z)-OBn (447 mg, 68% crude) as a fine yellow-white powder
1
was confirmed by H and 13C NMR and MS spectra.
According to general procedure E, Boc-Gly-Arg(NO2)-Ala-
(S-â-HAla-R-HB)2-Lys(Z)-OBn (300 mg, 0.26 mmol) was dis-
solved in TFE/CH3COOH (3:1, 4 mL) and hydrogenated in the
presence of Pd/BaSO4 (10%, 60 mg). The resulting precipitate
was purified by HPLC C8 (5-40% B, 30 min), tR 12.5 min, to
give after lyophilization the pure compound 27 in about 40%
1
yield. H NMR (300 MHz, D2O): δ 5.24-5.10 (m, 2H, CHO),
4.34-4.28 (m, 2H, CHN), 4.22-4.15 (m, 3H, CHN), 3.84 (s,
2H, CH2N), 3.19 (t, J ) 6.8, 2H, CH2NHC), 3.00-2.95 (m, 2H,
CH2NH2), 2.58-2.42 (m, 8H, CH2CHO, CH2CHN), 1.92-1.58
(m, 8H, CH2), 1.50-1.40 (m, 2H, CH2), 1.32 (d, J ) 7.5, 3H,
Me), 1.26 (d, J ) 6.2, 3H, Me), 1.25 (d, J ) 6.2, 3H, Me), 1.18-
1.13 (m, 6H, Me). FAB-MS: m/z 811 {10, (M + K)+}, 795 {32,
(M + Na)+}, 773 {100, (M + 1)+}. Purity by analytical HPLC
(0-100% B, 60 min, tR 18.7 min) >99%.
TF A•H-Ar g(NO2)-Ala -(R-â-HAla )4-Lys(Z)-OBn (26a ). Ac-
cording to general procedure B, to a solution in DMF (9 mL)
of the TFA salt 23a (1 equiv, 0.77 mmol) was added DIEA (0.53
mL, 3.08 mmol). HOBt (130 mg, 0.96 mmol), Boc-Arg(NO2)-
OH (295 mg, 0.92 mmol) and then EDC (183 mg, 0.96 mmol)
were successively added to the reaction. The resulting pre-
cipitate was dried under high vacuum and used without
further purification as it is not soluble in any solvent to be
purified. The presence of the desired Boc-Arg(NO2)-Ala-(R-â-
H-Gly-Ar g-Ala -(R-HB)2-(S-â-HAla )2-Lys-OH (28). Accord-
ing to general procedure D, to a solution in DMF (5 mL) of
the TFA salt 25 (1 equiv, 0.37 mmol) was added DIEA (0.19
mL, 1.11 mmol). HOBt (62 mg, 0.46 mmol), Boc-Gly-OH (77
mg, 0.44 mmol), and then EDC (88 mg, 0.46 mmol) were
successively added to the reaction. The resulting precipitate
was dried under high vacuum and used without further
purification as it is not soluble in any flash chromatography
or HPLC solvent. The presence of the desired Boc-Gly-Arg-
(NO2)-Ala-(R-HB)2-(S-â-HAla)2-Lys(Z)-OBn (252 mg, 60% crude)
1
HAla)4-Lys(Z)-OBn (757 mg, 81% crude) was confirmed by H
and 13C NMR and MS spectra. Further treatment with TFA
(2.7 mL), according to the general procedure D, gave the TFA
salt 26a , which was used without further purification.
TFA•H-Ar g(NO2)-(S-â-HAla-R-â-HAla)2-Lys(Z)-OBn (26b).
According to general procedure B, to a solution in DMF (9 mL)
of the TFA salt 23b (1 equiv, 0.83 mmol) was added DIEA
(0.057 mL, 0.33 mmol). HOBt (14 mg, 0.104 mmol), Boc-Arg-
(NO2)-OH (32 mg, 0.099 mmol), and then EDC (20 mg, 0.104
mmol) were successively added to the reaction. The resulting
precipitate was dried under high vacuum and used without
further purification as it is not soluble in any flash chroma-
1
was confirmed by H and 13C NMR and MS spectra.
According to general procedure E, Boc-Gly-Arg(NO2)-Ala-
(R-HB)2-(S-â-HAla)2-Lys(Z)-OBn (200 mg, 0.17 mmol) was
dissolved in TFE/CH3COOH (3:1, 3.5 mL) and hydrogenated
in the presence of Pd/BaSO4 (10%, 40 mg). The resulting

2328 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Poenaru et al.
precipitate was purified by HPLC (10-40% B, 30 min), tR 6.2
min, to give after lyophilization the pure compound 28 in about
25% yield. 1H NMR (300 MHz, D2O): δ 5.22-5.06 (m, 2H,
CHO), 4.28-4.20 (m, 3H, CHN), 4.16-4.04 (m, 2H, CHN), 3.76
(s, 2H, CH2N), 3.16-3.10 (m, 2H, CH2NHC), 2.93-2.86 (m,
2H, CH2NH2), 2.65-2.49 (m, 2H, CH2CHO), 2.43-2.32 (m, 2H,
CH2CHO), 2.36 (d, J ) 7.2, 2H, CH2CHN), 2.26 (d, J ) 7.2,
2H, CH2CHN), 1.85-1.53 (m, 8H, CH2), 1.41-1.28 (m, 2H,
CH2), 1.35 (d, J ) 7.2, 3H, Me), 1.20-1.14 (m, 6H, Me), 1.08-
1.03 (m, 6H, Me). FAB-MS: m/z 811 {12, (M + K)+}, 795 {23,
(M + Na)+}, 773 {100, (M + 1)+}. Purity by analytical HPLC
(0-100% B, 60 min, tR 19.5 min) >99%.
H-Gly-Ar g-Ala -(R-â-HAla )4-Lys-OH (29a ). According to
general procedure B, to a solution in DMF (7 mL) of the TFA
salt 26a (1 equiv, 0.68 mmol) was added DIEA (0.47 mL, 2.71
mmol). HOBt (115 mg, 0.85 mmol), Boc-Gly-OH (143 mg, 0.82
mmol), and then EDC (163 mg, 0.85 mmol) were successively
added to the reaction. The precipitate was dried under high
vacuum and used without further purification as it is not
soluble in any flash chromatography or HPLC solvent. The
presence of the desired Boc-Gly-Arg(NO2)-Ala-(R-â-HAla)4-Lys-
(Z)-OBn (622 mg, 80% crude) was confirmed by 1H and 13C
NMR and MS spectra.
According to general procedure E, Boc-Gly-Arg(NO2)-Ala-
(S-â-HAla)4-Lys(Z)-OBn (340 mg, 0.30 mmol) was dissolved in
TFE/CH3COOH (3:1, 4 mL) and hydrogenated in the presence
of Pd/BaSO4 (10%, 60 mg). The resulting precipitate was
purified by HPLC (10-40% B, 30 min), tR 6.8 min, to give after
1
lyophilization the pure compound 29c in about 30% yield. H
NMR (300 MHz, D2O): δ 4.34-4.27 (m, 2H, CHN), 4.24-4.09
(m, 5H, CHN), 3.84-3.81 (m, 2H, CH2N), 3.21-3.15 (m, 2H,
CH2NHC), 2.99-2.93 (m, 2H, CH2NH2), 2.45-2.41 (m, 2H,
CH2C9HN), 2.40-2.26 (m, 6H, CH2CHN), 1.92-1.57 (m, 8H,
CH2), 1.47-1.35 (m, 2H, CH2), 1.31 (d, J ) 7.2, 3H, Me), 1.15-
1.09 (m, 12H, Me). 13C NMR (75 MHz, D2O): δ 178.59, 176.58,
176.50, 176.08, 175.30, 170.09, 159.82, 56.22, 55.30, 52.71,
46.25, 45.22, 44.78, 43.37, 43.13, 42.00, 32.78, 31.13, 28.99,
27.02, 24.85, 22.16, 21.97, 19.50. FAB-MS: m/z 1542 {17, (2M
+ 2)+}, 771 {100, (M + 1)+}. Purity by analytical HPLC (0-
100% B, 60 min, tR 15.3 min) >99%.
H -Gly-Ar g-Ala -(R-â-H Ala )2-(S-â-H Ala )2-Lys-OH (29d ).
According to general procedure B, to a solution in DMF (5 mL)
of the TFA salt 26d (1 equiv, 0.42 mmol) was added DIEA
(0.29 mL, 1.68 mmol. HOBt (71 mg, 0.52 mmol), Boc-Gly-OH
(88 mg, 0.50 mmol), and then EDC (100 mg, 0.52 mmol) were
successively added to the reaction. The resulting precipitate
was dried under high vacuum and used without further
purification as it is not soluble in any flash chromatography
or HPLC solvent. The presence of the desired Boc-Gly-Arg-
(NO2)-Ala-(R-â-HAla)2-(S-â-HAla)2-Lys(Z)-OBn (333 mg, 70%
According to general procedure E, Boc-Gly-Arg(NO2)-Ala-
(R-â-HAla)4-Lys(Z)-OBn (200 mg, 0.18 mmol) was dissolved
in TFE/CH3COOH (3:1, 3.5 mL) and hydrogenated in the
presence of Pd/BaSO4 (10%, 40 mg). The resulting precipitate
was purified by HPLC (5-40% B, 30 min), tR 17.20 min, to
give after lyophilization the pure compound 29a in about 25%
1
crude) was confirmed by H and 13C NMR and MS spectra.
According to general procedure E, Boc-Gly-Arg(NO2)-Ala-
(R-â-HAla)2-(S-â-HAla)2-Lys(Z)-OBn (150 mg, 0.13 mmol) was
dissolved in TFE/CH3COOH (3:1, 3 mL) and hydrogenated in
the presence of Pd/BaSO4 (10%, 25 mg). The resulting pre-
cipitate was purified by HPLC (2-40% B, 30 min), tR 20.5 min,
to give after lyophilization the pure compound 29d in about
20% yield. 1H NMR (300 MHz, D2O): δ 4.36-4.28 (m, 2H,
CHN), 4.27-4.12 (m, 5H, CHN), 3.87-3.83 (m, 2H, CH2N),
3.23-3.18 (m, 2H, CH2NHC), 3.01-2.96 (m, 2H, CH2NH2),
2.346-2.43 (m, 2H, CH2CHN), 2.41-2.31 (m, 6H, CH2CHN),
1.92-1.60 (m, 8H, CH2), 1.50-1.40 (m, 2H, CH2), 1.35 (d, J )
7.5, 3H, Me), 1.17-1.11 (m, 12H, Me). FAB-MS: m/z 793 {15,
(M + Na)+}, 771 {100, (M + 1)+}. Purity by analytical HPLC
(0-100% B, 60 min, tR 15.4 min) >80%.
Molecu la r Dyn a m ics Sim u la tion s. Molecular mechanics
and dynamics calculations were realized using the AMBER
5.0 package66 using the parm96 parameter set and an all-atom
force-field representation.67 Force-field parameters for the ester
bonds were taken from the literature.68 Atomic charges for the
new monomers (R-HB, S-â-HAla, R-â-HAla) were calculated
using the GAUSSIAN94 package69 and the HF/6-31G* basis
set, by fitting atom-centered charges to an ab initio electro-
static potential, using the RESP method.70 Initial coordinates
for the MHC-ligand complexes were obtained from the X-ray
structure of HLA-B*270556 (Protein Data Bank code 1hsa) as
previously described.33,34 The spacers were substituted for the
natural pentapeptide sequence using the SYBYL 6.3 modeling
package (TRIPOS Assoc., Inc., St. Louis, MO). From a starting
fully extended conformation, dihedral angles of the main chain
between P3 and P9 were modified in order to reproduce a
correct trans geometry for the newly introduced amide or ester
bonds. The ligand was first relaxed by 1000 steps of conjugate
gradient energy minimization while maintaining the protein
fixed. It was then submitted to a 100-ps Simulated annealing
(SA) protocol in order to sample the broadest possible confor-
mational space. Starting with random velocities assigned at
a temperature of 1000 K, the peptide was first coupled to a
heat bath at 1000 K using a temperature coupling constant
Tτ of 0.2 ps and then linearly cooled to 50 K for the next 50 ps
while strengthening Tτ to a value of 0.05 ps. During these 100
ps, no protein atom was allowed to move. As the simulated
annealing was performed in vacuo, a distance-dependent
dielectric function (ꢀ ) 4r) was used. A twin cutoff (10.0, 15.0
Å) was used to calculate nonbonded electrostatic interactions
at every minimization step and every nonbonded pair list
update (10 steps), respectively.
1
yield. H NMR (300 MHz, D2O): δ 4.32-4.24 (m, 2H, CHN),
4.21-4.07 (m, 5H, CHN), 3.85-3.80 (m, 2H, CH2N), 3.20-3.14
(m, 2H, CH2NHC), 2.98-2.92 (m, 2H, CH2NH2), 2.50-2.25 (m,
8H, CH2CHN), 1.90-1.58 (m, 8H, CH2), 1.47-1.36 (m, 2H,
CH2), 1.32 (d, J ) 7.2, 3H, Me), 1.14-1.09 (m, 12H, Me). 13C
NMR (75 MHz, D2O): δ 178.57, 176.08, 175.29, 56.37, 55.39,
52.77, 46.22, 46.11, 45.20, 43.36, 43.15, 41.95, 32.66, 31.13,
28.98, 27.05, 24.86, 22.00, 19.43. FAB-MS: m/z 1542 {9, (2M
+2)+}, 771 {100, (M + 1)+}. Purity by analytical HPLC (0-
100% B, 60 min, tR 16.4 min) >99%.
H-Gly-Ar g-Ala -(S-â-HAla -R-â-HAla )2-Lys-OH (29b). Ac-
cording to general procedure B, to a solution in DMF (2 mL)
of the TFA salt 26b (1 equiv, 0.068 mmol) was added DIEA
(0.046 mL, 0.28 mmol). HOBt (12 mg, 0.085 mmol), Boc-Gly-
OH (14 mg, 0.082 mmol), and then EDC (16 mg, 0.085 mmol)
were successively added to the reaction. The resulting pre-
cipitate was dried under high vacuum and used without
further purification as it is not soluble in any flash chroma-
tography or HPLC solvent. The presence of the desired Boc-
Gly-Arg(NO2)-(S-â-HAla-R-â-HAla)2-Lys(Z)-OBn (70 mg) was
confirmed by FAB-MS.
According to general procedure E, Boc-Gly-Arg(NO2)-(S-â-
HAla-R-â-HAla)2-Lys(Z)-OBn (70 mg, 0.06 mmol) was dis-
solved in TFE/CH3COOH (3:1, 1 mL) and hydrogenated in the
presence of Pd/BaSO4 (10%, 10 mg). The resulting precipitate
was purified by HPLC (5-40% A, 30 min), tR 8.6 min, to give
after lyophilization the pure compound 29b in about 25% yield.
1H NMR (300 MHz, D2O): δ 4.26-4.18 (m, 2H, CHN), 4.18-
4.04 (m, 5H, CHN), 3.76-3.73 (m, 2H, CH2N), 3.12-3.07 (m,
2H, CH2NHC), 2.91-2.85 (m, 2H, CH2NH2), 2.40-2.32 (m, 2H,
CH2CHN), 2.30-2.22 (m, 6H, CH2CHN), 1.85-1.50 (m, 8H,
CH2), 1.40-1.30 (m, 2H, CH2), 1.26-1.22 (m, 3H, Me), 1.09-
1.02 (m, 12H, Me). FAB-MS: m/z 771 (86, [M + 1]+). Purity
by analytical HPLC (0-100% B, 60 min, tR 15.4 min) >99%.
H-Gly-Ar g-Ala -(S-â-HAla )4-Lys-OH (29c). According to
general procedure B, to a solution in DMF (8 mL) of the TFA
salt 26c (1 equiv, 0.77 mmol) was added DIEA (0.53 mL, 3.1
mmol). HOBt (131 mg, 0.97 mmol), Boc-Gly-OH (163 mg, 0.93
mmol), and then EDC (185 mg, 0.97 mmol) were successively
added to the reaction. The resulting precipitate was dried
under high vacuum and used without further purification as
it is not soluble in any flash chromatography or HPLC solvent.
The presence of the desired Boc-Gly-Arg(NO2)-Ala-(S-â-HAla)4-
Lys(Z)-OBn (719 mg, 82% crude) was confirmed by 1H and 13C
NMR and MS spectra.

Nonapeptide Analogue Binding Affinity to MHC Protein
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2329
From the last SA conformer, 13 counterions (9 Na+ and 4
Cl- ions) were then placed at electrostatic minima to neutralize
the protein, using the CION routine of AMBER.66 It was then
solvated in a 10-Å thick TIP3P water shell. After the solvent
was minimized by 1000 steps of steepest descent, the solvent
(water and counterions) was equilibrated by 25-ps MD at 300
K. The solvent was minimized again, and the fully solvated
complex was finally relaxed by 1000 steps of steepest descent.
The obtained coordinates were then used as a starting point
for a 500-ps MD simulation at 300 K. To avoid large drifts
from the protein crystal structure, a weak positional harmonic
constraint of 0.05 kcal‚mol-1‚Å-1 was applied to backbone
atoms of B*2705. As the solvent was implicitly taken into
account, a constant dielectric function (ꢀ ) 1) was utilized. For
the whole trajectory, the same twin cutoff (10-15 Å) was used
for calculating nonbonded interactions, and the nonbonded pair
list was updated every 10 steps. The SHAKE algorithm was
used on hydrogens with a tolerance of 0.00025 Å, a time step
of 2 fs, and Berendsen temperature coupling with separate
coupling of solute and solvent atoms to the heat bath.
Coordinates, velocities, and energies were saved every 0.5 ps.
All computations were done using the parallel version of
AMBER5.0 implemented on a CRAY J 90 cluster and an
INTEL paragon machine. The analyses of molecular dynamics
trajectories were achieved using in-house routines and the
CARNAL module of AMBER.66
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Ep itop e Sta biliza tion Assa y. The quantitative assay used
was previously described.71 Briefly, RMA-S transfectants
expressing B*2705 were used. These are murine cells with
impaired TAP-mediated peptide transport and low surface
expression of (empty) class I MHC molecules, which can be
induced at 26 °C72 and stabilized at the cell surface through
binding of exogenously added ligands. These cells were incu-
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M. Ligands with C50 e 5 µM were considered to bind with high
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Ack n ow led gm en t. This work is supported by the
Schweizerischer Nationalfonds zur Fo¨rderung der Wis-
senschaftlichen Forschung (Project No. 31-45504.95)
and by Grant SAF97/0182 from the Spanish Plan
Nacional de I+D to J .A.L.C. D.S. thanks Novartis
Pharma (Basel) for continuous financial support to his
group and A. K. Beck, J . Schreiber, and S. Sigrist for
processing the manuscript. D.R. thanks the calculation
center of the ETH-Zu¨rich for allocation of computer time
on the CRAY J 90 and PARAGON supercomputers.
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