Synthesis of Amide Bond Isosteres Incorporated

Synthesis of Amide Bond Isosteres Incorporated in the Minimal
Glycopeptide Recognized by T Cells in a Mouse Model for Rheumatoid Arthritis
Ida Andersson
Master Thesis in Organic Chemistry, 20p
Umeå University 2005
Supervisors:
Jan Kihlberg, Anna Linusson & Tomas Gustafsson
Examiner:
Mikael Elofsson
Abstract
Susceptibility to collagen induced arthritis is associated with the mouse MHCII molecule H2Aq. The rat CII260-267 sequence has been identified as the minimal glycopeptide required
for binding to H-2Aq and for eliciting a proper T cell response. This master thesis describes
the synthesis and incorporation of Ala261-Gly262 amide bond isosteres in the backbone of
CII260-267. The modified glycopeptides should be used to further investigate the interactions
between the peptide-MHCII complex and the T cell receptor in addition to the development of
more stable mimetics of the natural epitope for immunization of mice.
A methylene amine isostere was successfully synthesized and then incorporated in the
glycopeptide using solid-phase synthesis and the Fmoc-protocol. A ketomethylene building
block was also accomplished but during the following glycopeptide synthesis ring closure
involving the keto group was encountered. The synthesis of the final (E)-alkene isostere was
mainly focused on improving the E:Z selectivity in the crucial Wittig reaction. However, a
TBDMS protecting group was found to be surprisingly unstable which prompted a change in
the protective group strategy.
H
N
O
N
H
O
Amide bond
H
N
N
H
H
N
O
O
Methylene amine
H
N
O
O
Ketomethylene
37
(E)-Alkene
Contents
1.
List of Abbreviations................................................................................................. 1
2.
Background............................................................................................................. 2
3.
Aim of Diploma Work ............................................................................................... 6
4.
Results and Discussion ............................................................................................. 7
5.
Conclusions............................................................................................................22
6.
Future Prospects.....................................................................................................23
7.
Experimental Section...............................................................................................24
8.
Acknowledgements .................................................................................................35
9.
References.............................................................................................................36
37
1. List of Abbreviations
Ala
APC
aq
Aq
n-BuLi
t-BuOH
Boc
CIA
CII
DBU
DCC
DIC
DIPEA
DMF
DMSO
Fmoc
Gal
Gln
Glu
Gly
HATU
Alanine
Antigen-presenting cell
Aqueous
H-2Aq
n-Butyllithium
tert-Butyl alcohol
tert-Butoxycarbonyl
Collagen induced arthritis
Type II collagen
1,8-Diazabicyclo[5.4.0]undec-7-ene
N,N´-Dicyclohexylcarbodiimide
N,N´-Diisopropylcarbodiimide
Diisopropylethylamine
Dimethylformamide
Dimethylsulfoxide
9-Fluorenylmethoxycarbonyl
Galactose
Glutamine
Glutamic acid
Glycine
O-(7-Azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate
7-Aza-1-hydroxybenzotriazole
1-Hydroxybenzotriazole
High-performance liquid chromatography
δ-Hydroxylysine
Isoleucine
Lysine
Matrix assisted laser desorption/ionizationtime of flight
Methyl
Acetonitrile
Class II major histacompatibility complex
N-Methylmorpholine
Nuclear magnetic resonance
Phenylalanine
para-Methoxybenzyl
Proline
Rheumatoid arthritis
Room temperature
tert-Butyldimethylsilyl
tert-Butyldiphenylsilyl
T cell receptor
Trifluoroacetic acid
Triethylamine
Tetrahydrofuran
Thin layer chromatography
HOAt
HOBt
HPLC
Hyl
Ile
Lys
MALDI-TOF
Me
MeCN
MHCII
NMM
NMR
Phe
PMB
Pro
RA
rt
TBDMS
TBDPS
TCR
TFA
TEA
THF
TLC
371
2. Background
2.1. Antigen presentation involving class II MHC molecules
Antigen-presenting cells (APC) constitute an essential part of the immune system since they
take up and process extracellular protein antigens. This degradation results in shorter peptide
fragments that are bound to class II major histacompatibility complex (MHCII) molecules.
The peptide-MHCII complex is transported to the cell surface of the APC where it is
presented to helper CD4+ T cells (Figure 1). If the complex is recognized to be nonendogenous by the T cell receptor (TCR) then an immune response is initiated. This antigen
processing pathway does not only result in presentation of peptides derived from foreign
pathogens, such as bacteria, viruses and other microorganisms, instead most fragments
originate from endogenous proteins. Importantly, the immune system has certain mechanisms
that normally eliminate all T cells that recognize complexes of self peptide-MHCII in order to
maintain tolerance towards the host’s own tissues.1
Figure 1. The peptide–MHCII complex is transported to the cell surface of the APC where it is presented to
helper T cells for recognition. Adapted from Engelhard1.
2.2. Rheumatoid arthritis
An autoimmune disease is characterized by lack of tolerance displayed by the immune system
towards endogenous proteins. In rheumatoid arthritis (RA) this specifically leads to chronic
inflammations of the peripheral cartilaginous joints. Susceptibility to RA is associated with
certain human MHCII molecules, namely HLA-DR1 and HLA-DR4.2,3 This discovery
together with the observed organ-specificity of the inflammation indicate that a joint-specific
372
antigen is recognized by T cells. Type II collagen (CII), which is the main protein in joint
cartilage, has been proposed as a possible autoantigen.4
2.3. Collagen induced arthritis
A common animal model used for studying the disease mechanisms of RA is collagen
induced arthritis (CIA) where mice are immunized with rat CII.5 This leads to development of
similar histopathology of the affected joints as seen in RA patients. Susceptibility to CIA is
associated with the mouse MHCII H-2Aq molecule6 (referred to as Aq) and the development is
linked to presentation of the immunodominant rat CII peptide sequence 256-2707 (CII256270). These observations have been the starting point for extensive investigations of the
interactions between the peptide epitope, the Aq molecule and the receptors on CII specific T
cells. The studies have been based on a panel of twenty-nine T cell hybridomas obtained from
rat CII immunized mice expressing Aq on their antigen-presenting cells.8
T cell receptor (TCR)
HO
OH
O
HO
Gly256-Glu-Pro-Gly
H
N 260
O
Ala-Gly
H
N 263
OH
O
P1 pocket
N
H
O
NH2
264
Gly-Glu-Gln-Gly-Lys270
O
P4 pocket
H-2Aq molecule
Figure 2. Schematic representation of the immunodominant CII256-270 epitope anchored to the Aq protein and
interacting with the TCR. Adapted with small modifications from Holm et al9.
Alanine substitution of the amino acid residues in the CII256-270 epitope revealed that the
side chains of Ile260 and Phe263 anchored the peptide in the Aq binding site (Figure 2).10,11 In
addition, the two residues Lys264 and Glu266 were identified as major TCR contact points. A
majority of the T cell hybridomas (20 out of 29) specifically recognized CII256-270 with
Lys264 posttranslationally hydroxylated and glycosylated with a β-D-galactopyranosyl residue
(Figure 2).8 Four different groups of T cells could then be established based on the fine
specificity for individual hydroxyl groups on the carbohydrate moiety.12 Truncation of the
immunodominant epitope eventually identified the shorter CII260-267 sequence 1 as the
minimal glycopeptide required for binding to the Aq molecule and for eliciting a proper T cell
373
response (Figure 3).9 A methylene ether isostere of Ile260-Ala261 was incorporated in 1 to give
a modified glycopeptide that was found to have a lower affinity for the Aq molecule but was
still recognized by some T cells even though higher concentrations were required.13,14
HO OH
O
HO
O
NH2
OH
Gly-Glu-Gln267-NH2
Ac-Ile260-Ala-Gly-Phe N
H
O
1
Figure 3. The rat CII260-267 sequence 1 is the minimal glycopeptide that binds to Aq and elicits a proper T
cell response.
It has been observed that vaccination of mice with the galactosylated CII256-270 peptide can
protect them from CIA, and those that still develop arthritis are not affected as severely.15
This implies a possibility of using glycopeptides in the treatment of RA since mice transgenic
for the RA associated human MHCII molecule HLA-DR4 and human CII also are susceptible
to CIA.16 In this case, the immunodominant epitope recognized by T cells is located between
residues 259-273, which is slightly shifted compared to the CII256-270 epitope found in CIA.
2.4. Design of peptidomimetics as ligands for MHCII proteins
Modified peptide ligands aimed at interacting with MHCII molecules are mainly designed to
create stable mimetics of natural epitopes for immunizations. The key motive for developing
nonpeptide analogues, i.e. peptidomimetics, is to circumvent many of the unfavorable
pharmacokinetic properties displayed by peptides such as sensitivity towards enzymatic
degradation and poor absorption.
MHCII proteins are heterodimers, i.e. they consist of two subunits, and the peptide-binding
domain is constructed by both the α- and the β-chain. The binding groove, which consists of a
plane of an eight-stranded β-pleated sheet with two parallel α-helices at opposite sides, can
accommodate peptide ligands up to 20 residues long since it is open at both ends (Figure 4).
Peptides are bound in an extended β-strand conformation and generally with high affinity.
This is achieved by an array of hydrogen bonds along the length of the peptide backbone
establishing a sequence-independent type of interaction that makes it possible for MHCII
proteins to present many different types of antigens. The binding-specificity of different
MHCII molecules is derived from a few anchor interactions in pockets along the groove that
can accommodate specific amino acid side chains.17,18
374
Figure 4. Ribbon diagram showing the general secondary structure of the peptide-binding domain of MHCII
proteins. The peptide ligand is bound in an extended conformation in the groove consisting of an eight-stranded
β-pleated sheet and two parallel α-helices.
Development of peptidomimetics can be based on isosteric replacement of the amide bonds in
the peptide backbone. The amide group is planar and rigid due to its partial double bond
character derived from the delocalization of the lone pair electrons on the nitrogen atom into
the carbonyl group. The amide bonds in the extended β-strand peptide are almost coplanar
while the amino acid side chains can be found alternating above and below the plane of the
backbone.19 This gives a backbone conformation that is characterized by torsion angles
around the Cα-N (φ) bond, the Cα-C (ψ) bond and the amide bond (ω) (Figure 5).20
O
φ
N
H
Ri
ψH
O
ω
Ri+1
N
O
Figure 5. The peptide backbone can be characterized by three torsion angles around the Cα-N (φ) bond, the CαC (ψ) bond and the amide bond (ω), respectively.
A diverse set of amide bonds isosteres20 has been developed to mimic certain physiochemical
properties of the peptide bond while altering others (Figure 6). Hence, isosteric replacement
of the amide bond with different mimetics can be used to modify the three-dimensional
geometry as well as the electronic properties, such as the dipole moment and the possibility to
hydrogen bond. Introduction of N-methylated amide bonds has, for example, been used to
investigate the hydrogen bonding network between the backbone of a peptide ligand and a
MHCII protein.21 After the establishment of important interactions, N-methylation of
nonessential backbone amides increased the stability towards degradation by proteases. Other
375
examples of amide bond isosteres that have been incorporated in MHCII ligands include
retro-inverso22, (E)-alkene23 and methylene amine analogues23.
O
O
O
N
Me
N-Alkyl
N
H
Ketomethylene
(E)-Alkene
Methylene amine
O
N
H
Hydroxyethylene
Carba
O
X
N
H
Ether, X = O
Thioether, X = S
Sulfonamide
X
S
OH
S
H
N
O
Thioamide
Ester, X = O
Thioester, X = S
Retro-inverso
Figure 6. Amide bond isosteres.
3. Aim of Diploma Work
The aim of this diploma work was to replace one amide bond in the immunodominant type II
collagen glycopeptide 1 (Figure 7) with isosteres having different physicochemical properties.
The modified glycopeptides would be used to further investigate the requirements for the
minimal epitope to bind to the MHCII H-2Aq protein and for recognition by the T cell
hybridomas. Incorporation of amide bond isosteres in the peptide backbone would also confer
increased stability towards in vivo degradation, which is desirable if the peptide is to be used
for immunization of mice. The amide bond subjected to isosteric replacement was to be
chosen based on the hydrogen bonding network between the extended backbone of
glycopeptide 1 and the Aq protein. This would be accomplished by virtually docking the
glycopeptide into the binding site of a comparative model of Aq.
HO OH
O
HO
O
NH2
OH
Gly-Glu-Gln267-NH2
Ac-Ile260-Ala-Gly-Phe N
H
O
1
Figure 7. The immunodominant CII glycopeptide 1 to be modified by introduction of amide bond isosteres.
376
4. Results and Discussion
4.1. Amide bond isosteres to incorporate in glycopeptide 1
Three isosteres to be incorporated in glycopeptide 1 were chosen to mimic different properties
of the amide bond. The ketomethylene (ψ[COCH2]) isostere has retained hydrogen bond
acceptor properties but lack donor possibilities and is also more flexible compared to the
amide bond (Figure 8). Free rotation around the C-N bond in the methylene amine
(ψ[CH2NH]) isostere also confers increased flexibility. The amino group is ionized at
physiological pH which can protect from enzymatic degradation. However, introducing a
charge in the binding site may also have implications for the binding affinity if positioned
unfavorably. The three-dimensional geometry, bond length, bond angle and rigidity of the
amide bond are best mimicked by the (E)-alkene (ψ[(E)-CH=CH]). This isostere does not
alter the overall conformation of the peptide but it lacks all hydrogen bonding properties.20
i+1
O
H
N
R
i
R
N
H
i+1
R
H
N
O
Amide bond
R
i
N
H
O
H
N
O
R
Methylene amine
i
i+1
R
i+1
R
H
N
O
Ketomethylene
R
i
O
(E)-Alkene
Figure 8. The amide bond and the selected isosteres shown as dipeptide fragments where the amino acid side
chains are represented by R.
4.2. Docking Study of CII260-267 and MHCII H-2Aq
The aim of the docking study was to provide a detailed description of the interactions between
the minimal glycopeptide epitope CII260-267 (1) and the Aq protein. The hydrogen bonding
network between the extended backbone of the peptide ligand and the MHCII molecule
would then serve as a basis for selecting the amide bond to be replaced by different isosteres.
Since there is no available crystal structure of the Aq protein, the CII260-267 peptide was
instead virtually docked into the binding site of a comparative model24 of Aq using GOLD25.
As described earlier, extensive biological data confirm that the side chains of Ile260 and Phe263
anchor the peptide ligand in pockets in the binding site of the Aq protein while the β-Dgalactosyl residue is pointing out of the groove making critical contact with the TCR. Thus, a
docked pose of epitope 1 was only considered successful if these requirements were fulfilled
along with an extended β-strand backbone conformation and minimized solvent exposure.
However, it proved difficult for the docking program to simultaneously place both anchor
377
residues in their respective pocket while maintaining an extended backbone. One of the
generated conformations had correctly identified the side chains of Ile260 and Phe263 as
anchoring residues, but the C-terminal end of the peptide was pointing out of the binding site
shielding the galactosyl moiety from contact with the TCR (Figure 9). Another conformation
displayed an extended backbone but in this case the N-terminal acetyl group was placed in the
anchoring pocket instead of the side chain of Ile (Figure 9). However, the backbone
coordinates of Gly262-Phe263 in the middle part of these two conformations were found to be
very similar. It was therefore concluded that the two poses could be manually combined at
that amide bond to afford a final conformation having all desired features (Figure 10).
Side chain of Ile260
Side chain of Ile260
Side chains of Phe263
Figure 9. The two overlapping conformations generated in the docking analysis that were combined to give the
final pose of glycopeptide 1. Both the side chain of Ile260 and Phe263 in the conformation colored red are placed
in their respective pocket, but the backbone is twisted resulting in that the C-terminal end is pointing out of the
binding site shielding the galactosyl moiety from contact with the TCR. The conformation colored white, on the
other hand, displays an extended backbone conformation but has placed the N-terminal acetyl group in the
anchoring pocket instead of the side chain of Ile.
An extensive hydrogen bonding network was found between the backbone of the docked
glycopeptide and the Aq protein (Figure 11). The amino acid sequence from Ile260 to GalHyl264 contain the most essential features, i.e. the anchoring residues and the major TCR
contact point, and hence it was considered interesting to incorporate the isostere in this part of
the peptide. Based on the interactions provided by the model and the synthetic chemistry for
obtaining the isosteres, the amide bond between Ala261 and Gly262 was finally chosen for
isosteric replacement. According to the model, the carbonyl oxygen in this amide bond was
involved in hydrogen bonding and this would give the opportunity to further refine the model
since not all isosteres would be able to retain this interaction. From a synthetic point of view,
it would also be less complex to have only one chiral center to consider during the synthesis
of the dipeptide building blocks incorporating the isosteres.
378
260
Ile
Galactose
moiety
Phe
263
Figure 10. The docked conformation of glycopeptide 1 in the binding groove of Aq showing the side chains of
Ile260 and Phe263 anchoring the peptide in the P1 and P4 pocket, respectively, while the galactose moiety is
pointing out of the binding site. The molecular surface of the Aq protein is colored yellow for exposed, brown for
hydrophobic and green for hydrophilic.
H
N
N
N
H
O
N
H
O
N
H
H
N
OH
H
H
N
O
O
N
H
O
OH
O
N
H
H
HO OH
O
HO
O
H
N
N
H
O
O
NH3
H
N
O
O
O
O
O
H
N
N
H
O
NH2
O
O
NH2
O
O
N
H
H
N
H
H
H
O
Figure 11. The hydrogen bonding interactions between the backbone of CII260-267 and amino acid residues
in the Aq protein.
4.3. Summary of docking results and introduction to upcoming synthetic work
The docking analysis was used to derive the position in the CII260-267 peptide to be replaced
with three isosteres mimicking different physiochemical properties of the amide bond. Thus,
the aim is to synthesize dipeptide mimetics of Ala261-Gly262 containing a methylene amine, a
ketomethylene and an (E)-alkene isosteres (Figure 12) suitably protected for incorporation in
glycopeptide 1 using solid-phase synthesis.
379
a)
b)
O
N
H
H
N
O
N
H
O
HO OH
O O
HO
O OH
NH2
OH
O
O
O
H
H
H
N
N
N
N
N
NH2
H
H
O
O
O
O
H
N
Methylene amine
H
N
Ketomethylene
N
H
O
O
O
NH2
H
N
(E)-Alkene
O
1
Figure 12. a) Glycopeptide 1 with the Ala261-Gly262 position to be modified highlighted. b) The chosen
isosteres shown as Ala-Gly dipeptide mimetics.
4.4. Synthesis of the methylene amine isostere of Ala-Gly
4.4.1. Retrosynthetic analysis
Synthesis of Ala-Gly peptidomimetic 2 in protected form requires control of one stereocenter,
namely the former α-position in the alanine equivalent. The retrosynthetic analysis of this
methylene amine isostere (Figure 13) reveals that the chirality could be derived from the
Fmoc-protected amino acid L -alanine (7). Thus, synthesis of key intermediate 3 could be
accomplished by reductive alkylation of the primary amine in tert-butyl protected glycine (4)
with Fmoc protected L-alaninal (5) derived from the protected amino alcohol L-alaninol (6).
Furthermore, the decision was made to protect the secondary amine in 3 with a tertbutoxycarbonyl group to avoid the risk of it acting as a nucleophile in the solid-phase peptide
synthesis. Deprotection would however occur simultaneously as the glycopeptide is cleaved
from the solid support under acidic conditions making this protective group strategy
compatible with the Fmoc-protocol26.
Boc O
N
FmocHN
OH
FmocHN
OH
O
7
O
O
3
2
FmocHN
H
N
FmocHN
H
FmocHN
OH
O
6
O
H2N
5
Figure 13. Retrosynthetic analysis of the methylene amine building block 2 in protected form.
37
10
O
4
4.4.2. Synthesis of the methylene amine isostere 2
The synthetic route towards the methylene amine isostere 2 started with Fmoc-protected Lalanine 7 (Scheme 1). The carboxylic acid functionality was first activated as the mixed
anhydride 8 by reaction with N-methylmorpholine and isobutyl chloroformate.27 Subsequent
treatment with sodium borohydride furnished Fmoc-protected L-alaninol (6) in a quantitative
yield. Compound 6 was used after workup without any further purification in the following
oxidation step. Since N-protected α-amino aldehydes can be susceptible to epimerization of
the α-stereocenter it was desirable to employ a mild oxidation method in the following
synthesis of Fmoc-protected L-alaninal (5). Dess-Martin periodinane has, compared to other
oxidizing methods such as Swern and TEMPO, proven to be superior in the preparation of
highly epimerizable N-protected α-amino aldehydes from their corresponding alcohols.28
Thus, oxidation using Dess-Martin periodinane in a 1:1 mixture of CH2Cl2:DMSO afforded
aldehyde 5 which was used immediately after workup. Since epimerization of the αstereocenter was reported to be ≤1% in the literature28, the enantiomeric excess of 5 was not
investigated. However, the secondary amine 3 obtained after the following reductive
alkylation was found to be optically active and the stereochemical purity of the isostere after
being incorporated in the CII peptide was later assured by NMR studies. Hence, tert-butyl
protected glycine was reacted with aldehyde 5 followed by treatment with sodium
triacetoxyborohydride as reducing agent to give secondary amine 3 in a modest yield (43%).
a
OH
FmocHN
O
FmocHN
O
O
7
b
O
FmocHN
O
6
8
H
FmocHN
d
FmocHN
O
5
H
N
e
O
O
Boc O
N
FmocHN
H
N
FmocHN
3
f
c
OH
O
. TFA
OH
9
OH
2
Scheme 1. Reagents and conditions: (a) Isobutyl chloroformate, NMM, THF, -20 °C; (b) NaBH4, THF, MeOH, 20 °C, quantitative yield; (c) Dess-Martin periodinane, CH2Cl2:DMSO 1:1, rt; (d) H-Gly-OtBu ⋅ HCl, NaBH(OAc)3,
CH2Cl2, MeOH, rt, 43% from 6; (e) TFA, CH2Cl2, rt; (f) Boc2O, DIPEA, CH2Cl2, rt, 89% from 3.
37
11
Complete deprotection of the carboxylic acid to afford 9 as a TFA salt required stirring
overnight in 30% TFA in CH2Cl2. Finally, the secondary amine was directly protected with a
tert-butoxycarbonyl group by reaction with di-tert-butyl dicarbonate and DIPEA. This
afforded carboxylic acid 2 (89% over two steps) orthogonally protected for solid-phase
glycopeptide synthesis.
4.5. Synthesis of the ketomethylene isostere of Ala-Gly
4.5.1. Retrosynthetic analysis
Retrosynthesis of the Fmoc-protected ketomethylene isostere 10 (Figure 14) reveals that the
Boc-protected methyl ester of L-alanine (14) and tert-butyl glyoxylate (13) constitute suitable
building blocks. The latter could be derived from tert-butyl acrylate (15) through oxidative
cleavage of the alkene. Alternatively, the aldehyde could also be obtained by cleavage of the diol
in L -tartaric acid (16) after synthesis of its corresponding di-tert-butyl ester. A HornerWadsworth-Emmons reaction between β-ketophosphonate 12 and aldehyde 13 would then afford
key intermediate 11. Final reduction of the double bond would furnish the desired building block
10 suitably protected for direct use in solid-phase peptide synthesis.
O
O
FmocHN
BocHN
OH
O
O
O
10
11
O
O
O
BocHN
O
OMe
BocHN
O
P OMe
OMe
15
O
O
H
O
O
14
12
13
OH
OH
HO
OH O
16
Figure 14. Retrosynthetic analysis of the ketomethylene building block 10 in protected form.
4.5.2. Synthesis of tert-butyl glyoxylate 13
It was first investigated whether aldehyde 13 could be synthesized from tert-butyl acrylate
(15) by ozonolytic cleavage of the double bond followed by reduction of the intermediate
ozonide in presence of polymer-bound triphenylphosphine (Scheme 2). However, attempts to
obtain the aldehyde failed, giving rise to complicated mixtures with NMR spectra where only
37
12
traces of the product could be detected. This encouraged a modification in the synthetic
strategy where instead L-tartaric acid (16) was protected as its corresponding di-tert-butyl
ester 17 (24%). This was accomplished by esterification of 16 with a tert-butyl dicyclohexyl
isourea derivative obtained by reacting dicyclohexylcarbodiimide with tert-butyl alcohol in
the presence of a catalytic amount of CuCl.29
O
O
a
O
15
O
H
O
O
13
OH
O
OH
HO
OH
16
O
b
OH
O
O
O
c
H
O
OH O
O
17
13
Scheme 2. Reagents and conditions: (a) O3, PS-Triphenylphosphine, CH2Cl2, -78 °C → rt, 0%; (b) DCC, t-BuOH,
CuCl, CH2Cl2, rt, 24%; (c) NaIO4, MeOH:H2O 2:1, rt → 0 °C.
Initially, the following oxidative cleavage of diol 17 using NaIO4 in MeOH:H2O to afford
tert-butyl glyoxylate 13 appeared to give rise to a fairly large amount of undefined byproducts as indicated by 1H NMR spectroscopy even though only one product could be
detected by TLC analysis. Since the following Horner-Wadsworth-Emmons reaction initially
was plagued by very poor yields it was considered necessary to get a quantitative cleavage of
the diol. The reaction was repeated several times with NaIO4 and was also attempted using
Pb(OAc)4 and Na2CO3 in toluene but all efforts resulted in similar NMR spectra. However, it
was eventually found that the reported cleavage of the structurally related dimethyl protected
1
L-tartaric acid also gave rise to complicated H NMR spectra due to the existence of hydrated
and non-hydrated forms of the aldehyde.30 It was therefore concluded that this was probably
also the case with the corresponding tert-butyl ester and that the cleavage in fact was
quantitative.
4.5.3. Completing the synthesis of the Fmoc-protected ketomethylene isostere 10
The synthesis of β-ketophosphonate 12 was accomplished in 91% yield by reacting Bocprotected L-alanine methyl ester (14) with an excess of lithium dimethyl methylphosphonate
(Scheme 3).31 The following Horner-Wadsworth-Emmons reaction between β ketophosphonate 12 and tert-butyl glyoxylate 13 would then afford key intermediate 11.
37
13
However, it had been reported that this reaction could be accompanied by racemization of the
α-stereocenter and formation of a rearrangement product when using two equivalents of
triethylamine and MeCN as solvent at room temperature.31 This had been the case for βketophosphonates bearing small alkyl groups, such as i-Bu, Me and Bn, with the only
difference of having a benzyl protected ester in the aldehyde instead of tert-butyl. To avoid
these problems, 12 was instead treated with the weaker base N-methylmorpholine in CH2Cl2
at 0 °C followed by the addition of tert-butyl glyoxylate. Unfortunately, these conditions gave
only very low yields of alkene 11 (22%) although exclusively as the E isomer.
O
BocHN
a
OMe
O
BocHN
O
P OMe
OMe
O
b or c
BocHN
d
O
O
14
12
O
BocHN
O
e
11
O
TFA . H2N
OH
f
O
FmocHN
OH
O
O
18
19
O
10
Scheme 3. Reagents and conditions: (a) CH3PO(OMe)2, n-BuLi, THF, -78 °C, 91%; (b) CHOCOO-t-Bu (13),
NMM, 4Å molecular sieves, CH2Cl2, 0 °C, 22%; (c) CHOCOO-t-Bu (13), TEA, LiCl, MeCN, 0 °C → rt, 97%;
(d) H2, Pd/C, EtOAc, rt, 81%; (e) TFA, CH2Cl2, rt; (f) FmocOSu, MeCN:Na2CO3 (10% aq) 1:1, rt, 79% from
18.
Knowing that the degree of epimerization would have to be investigated, one equivalent of
triethylamine was instead used as base in MeCN at 0 °C.29 This furnished alkene 11 in
excellent yield (97%) as an isomeric mixture of E:Z in the ratio 3:1 as determined by 1H NMR
spectroscopy. The two isomers were never separated since in the following step the mixture of
alkenes was subjected to Pd/C catalyzed hydrogenation using EtOAc as solvent and H2 gas at
atmospheric pressure. This afforded the corresponding saturated analogue 18 in 81% yield
and even though the reaction was left stirring overnight it was observed that 2 hours was
enough for completion. Simultaneous cleavage of the tert-butoxycarbonyl group and the tertbutyl ester by treating 18 with TFA:CH2Cl2 1:5 for 2 hours provided the TFA salt of 19. The
resulting amino group was immediately Fmoc-protected by reaction with FmocOSu in
MeCN:Na2CO3 (10% aq) 1:1 to afford target building block 10 (79% over two steps).
37
14
4.5.4. Investigation of the stereoselectivity in the Horner-Wadsworth-Emmons reaction
It was necessary to investigate the degree of epimerization in the Horner-Wadsworth-Emmons
reaction when using one equivalent triethylamine as base. Initially, the strategy was to use
chiral HPLC to determine the enantiomeric excess by analyzing the Fmoc-protected
ketomethylene isostere 10 and its corresponding racemate. However, when a suitable eluent
separating the two enantiomers proved difficult to find the strategy was changed. The aim was
instead to synthesize Mosher amides32 21 and 23 and use 1H and 19F NMR spectroscopy to
determine the diastereomeric excess. One advantage of using 19F spectroscopy is that
overlapping peaks are less common than for 1H spectra, which could make the interpretation
more straightforward. Thus, simultaneous deprotection of the amine and the carboxylic acid
functionalities in 18 was achieved using the same conditions as described earlier (Scheme 4).
Subsequent treatment with MeOH in the presence of trimethylsilyl chloride furnished methyl
ester 20. Conversion into the corresponding Mosher amide 21 was accomplished by reaction
with DIPEA and (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride. The same
synthetic sequence was then applied on racemate 22 which was obtained by treating 18 with
DBU in THF at 80 °C for 30 min by microwave irradiation.
HCl . H2N
O
OMe
O
a, b
O
BocHN
c
F3C Ph H
N
MeO
O
20
O
O
OMe
O
21
O
18
O
d
BocHN
O
O
22
e, f, g
F3C Ph H
N
MeO
O
O
OMe
O
23
Scheme 4. Reagents and conditions: (a) TFA, CH2Cl2, rt; (b) TMSCl, MeOH, rt; (c) (S)-MTPA-Cl, DIPEA,
CH2Cl2, rt; (d) DBU, THF, 80 °C; (e) TFA, CH2Cl2, rt; (f) TMSCl, MeOH, rt; (g) (S)-MTPA-Cl, DIPEA,
CH2Cl2, rt.
After purification by flash column chromatography, the 1H spectrum of 23 clearly displayed
two peaks with the same integral for the diastereomeric methyl groups (Figure 15).
Unfortunately, the 19F spectrum was dominated by three large peaks having the relative
integrals 1.00:0.76:1.00. It was investigated if one of the peaks could be derived from the acid
chloride or the carboxylic acid of the Mosher reagent but neither spectrum displayed peaks
with the same shift. However, it was concluded that since the integral ratio in the 1H spectrum
was 1:1 this should also be the case in the corresponding 19F spectrum and therefore the two
37
15
largest peaks were assigned to the diastereomeric mixture of 23. This 1H spectrum was then
compared to the corresponding spectrum of crude 21. However, interfering signals from
impurities made it impossible to determine the diastereomeric excess of the crude and 21 was
therefore filtered through a pad of silica with care so as to minimize any changes in the
isomeric ratio. The diastereoselectivity could then be established as >100:1 (wanted:unwanted
diastereomer) since the 1H spectrum of 21 displayed a selectivity of 137:1 while integration of
the peaks in the 19F spectrum gave 192:1. Thus, it was concluded that the stereochemical
center had not been epimerized to a significant extent during the Horner-Wadsworth-Emmons
reaction and building block 10 could therefore be used in the solid-phase peptide synthesis.
3
O
F3C Ph H
N
MeO
1
2
O H CH3
a)
OCH3
1
O
23
3
3
3
b)
2
3
O
F3C Ph H
N
MeO
1
2
O H CH3
OCH3
1
O
21
2
3
Figure 15. a)
19
F and 1H spectra for the diastereomeric mixture of 23. Two peaks with the same integral are
clearly displayed for the methyl group (1) in the 1H spectrum while the
19
F spectrum shows three large peaks
with the integral ratio 1.00:0.76:1.00. The two peaks (3) having the same integral were assigned to 23. b)
19
F
1
and H spectra of 21 with the signals from the methyl group (1) enlarged.
4.6. An attempt to synthesize a hydroxyethylene isostere of Ala-Gly
It was also attempted to synthesize the corresponding hydroxyethylene isostere 25 from the
Fmoc-protected ketomethylene building block 10 since this would be an attractive way to
obtain another amide bond isostere of dipeptide Ala-Gly (Scheme 5). However, reduction of
the keto functionality in 10 using sodium borohydride in MeOH at -78 °C induced ring
closure and lactone 24 was the only observed product. This conclusion was based on the fact
37
16
that no OH proton could be found in the 1H NMR spectrum and although having triethylamine
in the eluent when analyzing the product with TLC it did not end up at the baseline. The
synthesis of the hydroxyethylene isostere was not explored any further.
O
O
FmocHN
OH
a
O
OH
FmocHN
instead of
FmocHN
OH
O
O
10
24
25
Scheme 5. Reagents and conditions: (a) NaBH4, MeOH, -78 °C.
4.7. Synthesis towards the (E)-alkene isostere of Ala-Gly
4.7.1. Retrosynthetic analysis
The synthetic strategy for obtaining the (E)-alkene isostere 26 of Ala-Gly was developed by
Wiktelius et al33. Throughout the synthetic sequence, the carboxylic acid in 26 would be
masked as a tert-butyldimethylsilyl protected alcohol that in the very last steps could be
deprotected and oxidized to afford key intermediate 27 (Figure 16). The critical step in the
synthetic sequence would be to obtain high E selectivity of 28 in the Wittig reaction where the
dianion of phosphonium salt 30 was to be reacted with aldehyde 29, obtained from 1,3propanediol (31). The phosphonium salt could be synthesized from alcohol 32 derived from
FmocHN
F3C
OH
N
H
27
O
OH
32
F3C
PPh3 I
N
H
30
H
OTBDMS
29
33
Figure 16. Retrosynthetic analysis of the (E)-alkene building block 26 in protected form.
37
17
OTBDMS
O
OH
H2N
N
H
28
O
N
H
F3C
OH
26
F3C
O
O
O
O
HO
OH
31
L -alaninol.
The amine in 33 was to be protected as a trifluoroacetamide since the use of
carbamate protective groups results in cyclization during the synthesis of the phosphonium
salt.33
4.7.2. Synthesis of aldehyde 29 and phosphonium salt 30
The first step in the synthetic sequence towards the (E)-alkene isostere was monosilylation of
the symmetric diol 31 (Scheme 6). This was accomplished by deprotonating 31 with one
equivalent of sodium hydride in THF which resulted in the precipitation of the corresponding
monosodium salt.34 Subsequent addition of tert-butyldimethylsilyl chloride furnished
monoprotected alcohol 34 in 78% yield. Oxidation using Dess-Martin periodinane afforded
the corresponding aldehyde 29 in a modest yield of 58%. This could be explained by the
finding that the silyl protective group was unstable during purification by silica gel flash
chromatography (even when packing the column with triethylamine in the eluent) as well as
during storage under N2 at -20 °C. For this reason it was difficult to keep the aldehyde
completely pure, instead a few percent of decomposition products were present in the
following Wittig reaction.
HO
a
OH
HO
OTBDMS
31
33
H
OTBDMS
34
OH
H2N
O
b
O
c
F3C
N
H
OH
29
O
d or e
F3C
32
N
H
35 X = I
36 X = Br
X
O
f
F3C
N
H
PPh3 X
30 X = I
Scheme 6. Reagents and conditions: (a) NaH, TBDMSCl, THF, rt, 78%; (b) Dess-Martin periodinane, CH2Cl2, rt,
58%; (c) Trifluoroacetic anhydride, TEA, CH2Cl2, 0 °C, 78%; (d) I2, PPh3, imidazole, toluene, rt, 79%; (e) CBr4,
PPh3, MeCN, 0 °C → rt, 80%; (f) PPh3, toluene, reflux, quantitative.
Phosphonium salt 30 was prepared from L-alaninol 33 by first protecting the amino group as a
trifluoroacetyl amide in 78% yield by reaction with trifluoroacetic anhydride in the presence
of triethylamine. Alcohol 32 was then converted into its corresponding iodide by reaction
with iodine, triphenylphosphine and imidazole.35 This provided 35 in 79% yield which was
finally transformed into its corresponding phosphonium salt 30 by treatment with
triphenylphosphine in toluene at reflux. Unfortunately, small traces of what appeared to be
triphenylphosphine oxide (∼3.5%) could not be removed, neither by flash chromatography nor
37
18
trituration. Bromide 3 6 was also synthesized in 80% yield by the addition of
tetrabromomethane and triphenylphosphine to alcohol 32. This would give the opportunity to
explore the selectivity in the Wittig reaction with regard to the counter ion, i.e. if the
phosphonium salt having iodide as counter ion would give low selectivity then the reaction
could be tested with bromide instead.
4.7.3. Investigation of the Wittig reaction
The Wittig reaction was accomplished by addition of two equivalents of titrated nbutyllithium to phosphonium salt 30 at –78 °C to form the corresponding ylide and to lithiate
the amide nitrogen (Scheme 7). The dianion was then reacted with aldehyde 29 and the
mixture was allowed to slowly reach 0 °C in order to equilibrate the double bond to the E
isomer36. Somewhat disappointingly, the highest obtained E:Z selectivity for 28 was 1:1 as
determined by 1H NMR spectroscopy in a combined yield of 36%. Since the two isomers
could not be directly separated using flash chromatography there was an obvious need for
improving the selectivity. Factors contributing to the low selectivity may have been that
neither the phosphonium salt nor the aldehyde was completely pure. As triphenylphosphine
oxide is rather inert and its weight was compensated for with regard to the equivalents, the
instability of the aldehyde seemed to be posing a bigger problem.
O
F3C
PPh3 I
N
H
O
a, b
F3C
N
H
OTBDMS
30
28
Scheme 7. Reagents and conditions: (a) 2 eq. n-BuLi, THF, -78 °C → rt; (b) 29, THF, -78 °C → 0 °C, E:Z 1:1 in
a combined yield of 36% .
4.8. Synthesis of modified CII260-267 glycopeptides
The Alaψ[CH2NH]Gly building block 2 was successfully incorporated in the type II collagen
fragment CII260-267 to afford the modified glycopeptide 37 using solid-phase peptide
synthesis (Figure 17). The synthesis was performed under standard conditions using the Fmoc
protocol and a polystyrene resin grafted with polyethylene glycol spacers (Tentagel resin).
The C-terminus was protected as an amide by using the Rink linker and the N-terminus was
acetylated to avoid charged termini. The methylene amine isostere 2 was activated with
HATU and 2,4,6-collidine followed by coupling to the peptide in a minimal amount of dry
DMF during 24 h. Cleavage from the solid phase was accomplished by treatment with a
mixture of TFA:H2O:thioanisole:ethanedithiol (35:2:2:1) for 3 h at 40 °C. This also resulted
in cleavage of all acid-labile side chain protective groups, while leaving the O-acetyl groups
37
19
which were removed by treatment with 20 mM NaOMe in methanol. Final purification by
reversed-phase HPLC afforded glycopeptide 37 in an overall yield of 26% (corrected for 70%
peptide content). Glycopeptide 37 was characterized by MALDI-TOF mass spectrometry and
1
H NMR spectroscopy (Table 1).
HO OH
O
HO
O
NH2
OH
Ac-Ile
H
N
N
H
Phe
O
N
H
Gly-Glu-Gln-NH2
O
37
Figure 17. The modified CII260-267 peptide 37 incorporating a methylene amine isostere of Ala261-Gly262.
Table 1. 1H NMR chemical shifts for peptide 37 containing Ala261ψ[CH2NH2+] Gly262 recorded in water with
10% D2Oa.
Residue
HN (amide)
Ile260b
8.34
Ala261ψ[CH2NH2+] Gly262
Hα
4.28
Hβ
2.07
8.33 (Ala)
4.41 (Ala)
4.08 (Gly)
Phe263
8.76
4.93
1.42 (CH3)
3.21; 3.27
(CH2NH2+)
3.33; 3.20
Hyl264c
8.73
4.47
2.19; 1.97
Hγ
1.62; 1.39
Other
1.10 (γCH3)
1.06 (δCH3)
7.55 (3,5H)
7.52 (4H)
7.46 (2,6H)
1.81
3.38; 3.19 (εCH2)
7.82 (εNH3+)
4.22 (δH)
Gly265
8.33
4.15
Glu266
8.41
4.53
2.28; 2.09
2.63; 2.58
Gln267d
8.67
4.48
2.30; 2.18
2.55
7.68; 7.03 (δNH2)
a
Measured at 500 MHz and 298 K.
b
Chemical shift for the N-terminal acetate group: δ = 2.24.
c
Chemical shifts for the galactose moiety: δ = 4.63 (H1), 4.12 (H4), 3.97 (H6 and H6´), 3.90 (H5), 3.84 (H3)
and 3.73 (H2).
d
Chemical shifts for the C-terminal amide: δ = 7.78, 7.29.
Incorporation of the Alaψ[COCH2]Gly building block 10 in the type II collagen fragment
CII260-267 using solid-phase peptide synthesis proved to be more challenging. Use of the
same conditions as previously described gave two major peaks as determined by HPLC.
Separation of the two peaks using reversed-phase preparative HPLC and different gradients of
MeCN:H2O with 0.1% TFA proved unsuccessful but by changing the eluent to MeOH:H2O
with 0.1% TFA they were finally separated. Structural studies then revealed that the major
peak was not the desired glycopeptide 38 (Figure 18). Instead the ring closed analogue 39,
37
20
where the α-amine in isoleucine has made a nucleophilic attack on the ketone functionality in
the isostere, was the most probable major product having the correct mass even though all
signals could not be assigned using 1H NMR spectroscopy. The second largest peak turned
out to be a mixture of two glycopeptides, the minor one possibly being the desired
glycopeptide 38.
HO OH
O
HO
O
NH2
OH
Ac-Ile
H
N
HO OH
O
HO
O
O
Phe
O
N
H
Phe
O
O
38
NH2
N
HN
Gly-Glu-Gln-NH2
O
OH
N
H
Gly-Glu-Gln-NH2
O
39
Figure 18. The desired modified peptide 38 incorporating a ketomethylene isostere of Ala261-Gly262 and the
proposed ring closed analogue 39 obtained as the main product.
37
21
5. Conclusions
The aim of this diploma work was to incorporate three amide bond isosteres in the backbone
of the minimal glycopeptide epitope 1. The amide bond between Ala261-Gly262 was selected
based on a computational model visualizing the interactions between the peptide ligand and
the Aq protein, while also considering synthetic challenges imposed by different dipeptide
building blocks. Ala-Gly mimetics having a methylene amine (2), a ketomethylene (10) and
an (E)-alkene (26) isostere, respectively, were target molecules chosen to be incorporated in
the glycopeptide using Fmoc solid-phase peptide synthesis (Figure 19).
Boc O
N
FmocHN
O
OH
O
FmocHN
OH
FmocHN
OH
O
2
10
26
Figure 19. Ala-Gly peptidomimetics incorporating a methylene amine (2), a ketomethylene (10) and an (E)alkene (26) isostere as target building blocks.
The orthogonally protected methylene amine isostere 2 was successfully synthesized and
incorporated in the minimal epitope to afford the modified glycopeptide 37. Synthesis of the
ketomethylene isostere 10 included investigation of the stereoselectivity in the essential
Horner-Wadsworth-Emmons reaction by synthesis of Mosher amides. 1H and 19F NMR
spectroscopy was used to conclude that the obtained selectivity was >100:1. Incorporation of
dipeptide building block 10 in the glycopeptide sequence unfortunately resulted in ring
closure by nucleophilic attack of the α-amine in isoleucine on the ketone functionality in the
isostere. The (E)-alkene isostere 26 was the most synthetically challenging building block
since high E:Z selectivity proved difficult to obtain. The main effort was put on improving the
Wittig reaction but it was eventually realized that the protective group for the alcohol would
have to be changed in order to obtain a more stable aldehyde.
37
22
6. Future prospects
Synthesis of glycopeptide 38, incorporating the ketomethylene isostere, still remains to be
accomplished since the obtained main product most likely was the ring closed analogue 39.
The nucleophilic attack on the keto functionality of the free amine in isoleucine could be
avoided by coupling Ac-Ile-OH instead of the Fmoc-protected amino acid. The last Fmoc
deprotection and the following acetylation of the N-terminal are then avoided and instead the
amine is protected throughout the synthesis as an amide making the nitrogen considerably less
nucleophilic.
Some modifications have to be made in the synthetic sequence towards the (E)-alkene
building block 26 (Figure 20). Due to the instability of the TBDMS ether it is necessary to
change the protective group on the alcohol. Synthesis of the corresponding paramethoxybenzyl (PMB) ether or the tert-butyldiphenylsilyl (TBDPS) ether could be attractive
approaches since these protective groups are considerably more acid stable. Importantly, the
alkene functionality should not be affected during deprotection by treatment with DDQ and
TBAF, respectively. The Wittig reaction must also to be explored further to obtain higher E
selectivity and before the isostere can be incorporated in the glycopeptide it has to be oxidized
to the carboxylic acid and the protective group on the amine should be changed to an Fmoc
group. Finally, the modified glycopeptides incorporating the three amide bond isosteres
should be biologically tested for binding to the Aq protein and recognition by the T cell
hybridomas.
O
F3C
O
N
H
PPh3 X
O
F3C
H
OPg
Wittig
reaction
O
F3C
N
H
Oxidation followed by
N
H
OH
OPg
O
protective group
manipulation
OH
FmocHN
26
Figure 20. Remaining synthetic steps for obtaining the (E)-alkene isostere 26 of Ala-Gly.
37
23
Deprotection
7. Experimental Section
7.1. Comparative modeling24 of MHCII H-2Aq
A three-dimensional structure of H-2Aq was modeled based on a crystal structure of the
murine class II MHC I-Ab in complex with a human CLIP peptide (pdb code: 1MUJ)37. The
template, with a resolution of 2.15 Å, was chosen based on its high sequence identity with the
Aq protein (89% in the binding site). The quality of the template was first evaluated using
WHATCHECK38 followed by addition of hydrogens to the PDB molecular structure file by
using the program Reduce39. Water molecules were removed when importing the crystal
structure in MOE40 after which it was subjected to relaxation using the AMBER94 force field.
The minimization was terminated when the root mean square gradient was <10 using a
combination of three methods; steepest descent, conjugated gradient and truncated Newton,
respectively. The comparative modeling was then performed in MOE for the α- and β-chain
of Aq separately. Each chain was first aligned with its corresponding sequence in the template
using an iteration limit of 1000. Ten comparative models were built for each chain ignoring
any outgaps and applying medium minimization. The best intermediate model for each chain
according to the scoring function within MOE were then combined to afford the final
comparative model which was energy minimized with a gradient of 1.0 using the same
method as previously described.
7.2. Computational docking study of CII260-267 and H-2Aq
The initial three-dimensional atomic coordinates of the CII260-267 glycopeptide 1 were
generated from a SMILES string using CORINA software41. This conformation was docked
into the binding site of the refined comparative model of MHCII H-2Aq using Gold25. The
dockings were terminated after 30 genetic algorithm (GA) runs and the generated
conformations were evaluated using the fitness function GoldScore implemented in Gold. A
maximum number of 100 000 operations were performed for each GA run where the number
of islands were defined as 5, each with the population size of 100. The docking parameters
crossover, mutate and migrate were set to 95, 100 and 0, respectively, whereas niche size was
given the value 3. Furthermore, the parameters selection pressure, Van der Waals and
hydrogen bonding were set to 1.1, 10 and 5, respectively. The parameters were selected based
on previous unpublished results42. The radius of the active site was defined to be 17 Å from a
point with the coordinates (x,y,z) (49.118, 34.126, 78.910) found in the centre of the binding
site. Since biological results show that the galactose moiety points out of the binding site
making contact with the TCR, a constraint was applied where the galactose moiety on Hyl264
was forced to have a minimal separation of 10 Å from a point with the coordinates (x,y,z)
(39.898, 37.973, 75.635) on the surface of the large P4 pocket in the middle of the binding
37
24
site. A spring constant of 5 was applied; topology was accounted for and ligands were not
docked where constraints were physically impossible. The thirty highest ranked
conformations were subjected to visual inspection and manual ranking. The highest ranked
conformation (based on the following criteria: extended β-strand backbone conformation,
anchoring residues placed in pockets, sugar moiety pointing out of the binding site, minimized
solvent exposure) given as a solution from the first docking was energy minimized with the
Aq protein and then used as the input conformation in a new docking run. No conformation
from the second run fulfilled all desired criteria but two generated poses were found to
complement each other, meaning that they together fulfilled all requirements. Since the
backbone coordinates of Gly262-Phe263 in the two conformations were found to be very similar
it was concluded that they could be manually combined at that amide bond. This furnished the
final conformation of CII260-267 that was energy minimized with the H-2Aq protein using
the same method in MOE as previously described with AMBER94 force field and a gradient
of 0.05.
7.3. General synthetic procedures
All reactions were carried out under an inert N2 atmosphere with dry solvents under
anhydrous conditions, unless otherwise mentioned. THF was distilled from potassium while
CH2Cl2 and MeCN were distilled from calcium hydride. DMF was distilled under vacuum
and then stored for a short period of time over molecular sieves (4Å). MeOH was dried over
molecular sieves (3Å). TLC was performed on Silica Gel 60 F254 (Merck) with detection by
UV light and staining with PMA (a solution of ethanolic phosphomolybdic acid), Seebach
(phosphomolybdic acid and Ce(SO4)2.4H2O in aqueous H2SO4) or permanganate (KMnO4 and
K2CO3 in aqueous NaOH) followed by heating. Flash column chromatography was performed
on silica gel (Matrex, 60 Å, 35-70 µm, Grace Amicon). Analytical reversed-phase HPLC was
performed on a Beckman System Gold HPLC equipped with a Kromasil C-8 column
(250×4.6 mm) using a 0-100% linear gradient of MeCN (0.1% TFA) in H2O (0.1% TFA) over
60 min. A flow-rate of 1.5 mL/min was used and detection was at 214 nm. Preparative
reversed-phase HPLC was performed on a Kromasil C-8 column (250×20 mm) using the
same eluent, a flow-rate of 11 mL/min and detection at 214 nm. Specific rotations were
measured with a Perking-Elmer model 343 polarimeter. 1H and 13C NMR spectra were
recorded on a Bruker DRX-400 spectrometer at 400 MHz and 100 MHz, respectively, at 298
K and calibrated using the residual peak of solvent as internal reference (CDCl3 [CHCl3 δ H
7.27, CDCl3 δC 77.0 ppm] or CD3OD [CD2HOD δH 3.34, CD3OD δ C 49.0 ppm]. First-order
chemical shifts and coupling constants were obtained from one-dimensional spectra while
carbon and proton resonances were assigned from COSY and HETCOR experiments.
Aromatic Fmoc carbon signals have not been assigned. Spectra for glycopeptide 37 were
recorded in H2O:D2O 9:1 at 298 K on a Bruker AMX-500 spectrometer at 500 MHz.
37
25
((1S)-2-Hydroxy-1-methyl-ethyl)-carbamic acid 9H-fluoren-9-ylmethyl ester (6)
A solution of Fmoc-Ala-OH (2.00 g, 6.42 mmol) in THF (14 mL) was treated with Nmethylmorpholine (776 µL, 7.07 mmol) at –20 °C. After stirring for 5 min, isobutyl
chloroformate (922 µL, 7.07 mmol) was added dropwise. A second portion of isobutyl
chloroformate (160 µL, 1.23 mmol) was added after an additional 40 min followed by stirring
for 10 min. The white precipitate was filtered off and rinsed with dry THF (20 mL). To the
solution of mixed anhydride 8 was then NaBH4 (720 mg, 19.3 mmol) added in one portion at
–20 °C followed by careful addition of MeOH (41 mL). After stirring for 90 min, NH4Cl (aq,
sat.), water and EtOAc were added and the phases were separated. The water phase was
extracted with EtOAc three times and the combined organic layers were washed with NaCl
(aq, sat.), dried over Na2SO4 and filtered. Concentration then afforded crude alcohol 6 (1.87 g,
complete conversion of starting material) as a white solid, which was used without further
purification. 1H NMR (CDCl3): δ = 7.78 (d, J = 7.5 Hz, 2H, Fmoc-arom), 7.60 (d, J = 7.5 Hz,
2H, Fmoc-arom), 7.41 (t, J = 7.4 Hz, 2H, Fmoc-arom), 7.33 (t, J = 7.4 Hz, 2H, Fmoc-arom),
4.88 (brs, 1H, NH), 4.44 (d, J = 5.6 Hz, 2H, Fmoc-CH2), 4.22 (t, J = 6.5 Hz, 1H, Fmoc-CH),
3.91-3.78 (m, 1H, CH), 3.72-3.62 (m, 1H, CH2), 3.59-3.49 (m, 1H, CH2), 1.19 (d, J = 5.6 Hz,
3H, CH3); 13C NMR (CDCl3): δ = 156.6 (NCO), 143.9, 143.9, 141.3, 127.7, 127.0, 127.0,
125.0, 125.0, 120.0, 66.9 (CH2), 66.7 (Fmoc-CH2), 49.0 (CH), 47.3 (Fmoc-CH), 17.3 (CH3).
((1S)-1-Methyl-2-oxo-ethyl)-carbamic acid 9H-fluoren-9-ylmethyl ester (5)
Alcohol 6 (800 mg, 2.69 mmol) dissolved in CH2Cl2:DMSO (1:1, 24 mL) was treated with
Dess-Martin periodinane (9.50 ml, 4.57 mmol, 15 wt% solution in CH2Cl2) followed by
stirring for 40 min at room temperature. The reaction was quenched by addition of Na2S2O5
(5.37 g, 28.2 mmol) dissolved in NaHCO3 (aq, sat.). The phases were separated and the
organic layer was washed with NaHCO3 (aq, sat.) and H2O. The combined water phases were
extracted with CH2Cl2 and the combined organic layers were finally washed with NaCl (aq,
sat.), dried over Na2SO4 and filtered. Concentration under cold conditions afforded crude
aldehyde 5 (643 mg) as a white solid, which was used immediately without further
purification. 1H NMR (CDCl3): δ = 9.57 (s, 1H, CHO), 7.78 (d, J = 7.5 Hz, 2H, Fmoc-arom),
7.61 (d, J = 7.1 Hz, 2H, Fmoc-arom), 7.42 (t, J = 7.5 Hz, 2H, Fmoc-arom), 7.33 (dt, J = 1.2,
7.5 Hz, 2H, Fmoc-arom), 5.43 (brs, 1H, NH), 4.50-4.40 (m, 2H, Fmoc-CH2), 4.33 (quin, J =
7.2 Hz, 1H, CH), 4.24 (t, J = 6.7 Hz, 1H, Fmoc-CH), 1.39 (d, J = 7.3 Hz, 3H, CH3); 13C NMR
(CDCl3): δ = 198.9 (CHO), 155.8 (NCO), 143.7, 143.7, 141.3, 127.7, 127.7, 127.1, 125.0,
120.0, 67.0 (Fmoc-CH2), 55.9 (CH), 47.1 (Fmoc-CH), 14.8 (CH3).
[(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-propylamino]-acetic acid tert-butyl ester (3)
Glycine tert-butyl ester hydrochloride (438 mg, 2.61 mmol) was added to a solution of crude
aldehyde 5 (643 mg, 2.18 mmol) in CH2Cl2 (15 mL) followed by stirring for 40 min at room
37
26
temperature. NaBH(OAc)3 (738 mg, 3.48 mmol) and MeOH (3.6 mL) were added followed
by stirring for 90 min and then the reaction was quenched by addition of NaHCO3 (aq, sat.).
The organic phase was washed with NaCl (aq, sat.) and the water phase was then extracted
with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and concentrated.
Purification by flash column chromatography (n-heptane:EtOAc:HOAc 20:30:1 → nheptane:EtOAc:TEA 10:40:1) afforded secondary amine 3 (468 mg, 43% from 6) as a slightly
yellow oil. [α]D +2.1 (c 1.0, CHCl3); 1H NMR (CDCl3): δ = 7.77 (d, J = 7.5 Hz, 2H, Fmocarom), 7.63 (d, J = 7.5 Hz, 2H, Fmoc-arom), 7.40 (t, J = 7.4 Hz, 2H, Fmoc-arom), 7.32 (dt, J
= 0.8, 7.4 Hz, 2H, Fmoc-arom), 5.26 (brs, 1H, CONH), 4.40 (d, J = 5.4 Hz, 2H, Fmoc-CH2),
4.24 (t, J = 6.9 Hz, 1H, Fmoc-CH), 3.81 (brs, 1H, CH), 3.34 (d, J = 17.4 Hz, 1H, COCH2),
3.28 (d, J = 17.4 Hz, 1H, COCH2), 2.67 (brs, 2H, NCH2CH), 1.97 (brs, 1H, NH), 1.49 (s, 9H,
C(CH3)3), 1.19 (d, J = 3.3 Hz, 3H, CH3); 13C NMR (CDCl3): δ = 171.7 (COO), 156.1 (NCO),
144.0, 144.0, 141.3, 127.6, 127.0, 125.1, 119.9, 81.3 (C(CH3)3), 66.5 (Fmoc-CH2), 54.2
(NCH2CH), 51.6 (COCH2), 47.3 (Fmoc-CH), 46.8 (NCH), 28.1 (C(CH3)3), 18.9 (CH3).
{tert-Butoxycarbonyl-[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propyl]-amino}-acetic
acid (2)
Secondary amine 3 (255 mg, 0.621 mmol) dissolved in CH2Cl2 (20 mL) was treated with TFA
(8.6 mL) followed by stirring overnight (14 h) at room temperature. The solution was
concentrated and then redissolved and concentrated from CHCl3 three times to remove
residual TFA. To the TFA salt of 9 dissolved in CH2Cl2 (12 mL) was DIPEA (238 µL, 1.37
mmol) added dropwise followed by addition of Boc2O (186 mg, 0.853 mmol) dissolved in
CH2Cl2 (2.0 mL). After stirring for 40 min, a second portion of DIPEA (11 µL, 0.063 mmol)
and Boc2O (104 mg, 0.477 mmol) was added. A third portion of DIPEA (55 µL, 0.313 mmol)
and Boc2O (73 mg, 0.334 mmol) was added after 30 min and the solution was then stirred an
additional 15 min. The reaction was quenched by addition of citric acid (10%, aq), the phases
were separated and the water phase was extracted with CH2Cl2. The combined organic layers
were washed with NaCl (aq, sat.), dried over Na2SO4, filtered and concentrated. Purification
by flash column chromatography (n-heptane:EtOAc:HOAc 75:25:2 → 33:17:1) gave
carboxylic acid 2 (252 mg, 89%) as a white foam. [α] D –5.1 (c 1.0, CHCl3); 1H NMR
(CDCl3), rotamers: δ = 7.76 (d, J = 7.5 Hz, 2H, Fmoc-arom), 7.63-7.54 (m, 2H, Fmoc-arom),
7.39 (t, J = 7.5 Hz, 2H, Fmoc-arom), 7.35-7.26 (m, 2H, Fmoc-arom), 5.56 (d, J = 6.7 Hz,
0.6H, NH), 5.08 (d, J = 6.3 Hz, 0.4H, NH), 4.42 (d, J = 5.4 Hz, 0.8H, Fmoc-CH2), 4.33 (d, J =
6.8 Hz, 1.2H, Fmoc-CH2), 4.24-4.15 (m, 1H, Fmoc-CH), 4.03-3.92 (m, 2H, CH2CO2H), 3.923.80 (m, 0.8H, CH), 3.72-3.59 (m, 0.7H, NCH2), 3.59-3.48 (m, 0.2H, CH), 3.44-3.34 (m,
0.3H, NCH2), 3.22 (d, J = 14.3 Hz, 0.4H, NCH2), 3.06 (dd, J = 2.9, 14.4 Hz, 0.7H, NCH2),
1.45 (s, 3.2H, C(CH3)3), 1.42 (s, 5.8H, C(CH3)3), 1.18 (d, J = 5.1 Hz, 2.5H, CHCH3), 1.02
(brs, 0.5H, CHCH3); 13C NMR (CDCl3), rotamers [δ for minor rotamer when assigned]: δ =
37
27
174.2 [173.9] (COOH), 156.5 [156.3] (NCOOCH2), 155.9 (NCOOC(CH3)3), 143.9 [143.8],
141.3 [141.2], 127.7, 127.0, 125.2 [124.9], 119.9 [119.9], 81.2 [81.5] (C(CH3)3), 66.8 [66.5]
(Fmoc-CH2), 52.8 [53.3] (NCH2CH), 49.8 (NCH 2COOH), 47.2 (Fmoc-CH), 46.9 [46.7]
(NCH), 28.2 [28.1] (C(CH3)3), 18.6 (CH3).
(2R,3R)-Di-tert-butyl tartrate (17)
N,N´-Dicyclohexylcarbodiimide (20.0 g, 96.9 mmol), tert-butyl alcohol (11.2 mL, 118 mmol)
and CuCl (400 mg, 4.04 mmol) were stirred for 5 days at room temperature. CH2Cl2 (140 mL)
and L-tartaric acid (4.82 g, 32.1 mmol) were added followed by continued stirring for 48 h.
The solution was first filtered through a pad of Celite and then washed with H2O, dried over
Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography
(n-heptane:EtOAc 5:1 → 4:1) to afford di-tert-butyl protected tartaric acid 17 (2.00 g, 24%)
as a white solid. [α]D +5.9 (c 1.0, CHCl3); 1H NMR (CDCl3): δ = 4.37 (dd, J = 1.2, 6.0 Hz,
2H, CH), 3.13 (dd, J = 1.2, 6.0 Hz, 2H, OH), 1.52 (s, 18H, CH3); 13C NMR (CDCl3): δ =
170.9 (CO), 83.4 (C(CH3)3), 72.3 (CH), 28.0 (CH3).
tert-Butyl glyoxylate (13)
A solution of NaIO4 (1.17 g, 5.47 mmol) in H2O (8.0 mL) was added to diol 17 (710 mg, 2.71
mmol) dissolved in MeOH (16 mL) at room temperature. The solution was cooled to 0 °C
followed by stirring for 2 h. Et2O and H2O were added, the phases were separated and the
water phase was extracted with Et2O. The combined organic layers were dried over Na2SO4
and filtered. Concentration under cold conditions gave crude aldehyde 13 as a colorless oil of
low viscosity, which was used immediately without further purification.
(3S)-(3-tert-Butoxycarbonylamino-2-oxo-butyl)-phosphonic acid dimethyl ester (12)
n-BuLi (12.3 mL, 19.7 mmol, 1.6 M in hexanes) was slowly added to dimethyl
methylphosponate (2.10 ml, 19.7 mmol) dissolved in THF (18 mL) at -78 °C. After stirring
for 25 min, a solution of Boc-Ala-OMe (500 mg, 2.46 mmol) in THF (12 mL) was added
dropwise. The cooling bath was removed after 60 min followed by additional stirring for 30
min. The reaction was quenched by addition of citric acid (10% aq), the phases were
separated and the water phase was extracted with EtOAc. The combined organic layers were
washed with NaCl (aq, sat.), dried over Na2SO4 and filtered. Concentration followed by
purification by flash column chromatography (n-heptane:EtOAc 1:3 → 1:6) afforded βketophosphonate 12 (658 mg, 91%) as a colorless oil. [α]D –1.4 (c 1.0, CHCl3); 1H NMR
(CDCl3): δ = 5.41 (d, J = 6.7 Hz, 1H, NH), 4.35 (quin, J = 7.2 Hz, 1H, CH), 3.80 (d, J = 2.5
Hz, 3H, OCH3), 3.77 (d, J = 2.5 Hz, 3H, OCH3), 3.30 (dd, J = 14.2, 22.6 Hz, 1H, CH2), 3.12
(dd, J = 14.2, 22.6 Hz, 1H, CH2), 1.43 (s, 9H, C(CH3)), 1.34 (d, J = 7.2 Hz, 3H, CH3); 13C
NMR (CDCl3): δ = 201.8 (J = 6.4 Hz, CO), 155.2 (NCO), 80.0 (C(CH3)3), 56.0 (CH), 53.1 (J
37
28
= 2.7 Hz, OCH3), 53.1 (J = 2.7 Hz, OCH 3), 37.6 (J = 131 Hz, CH2), 28.3 (C(CH3)3), 16.9
(CH3).
(5S)-5-tert-Butoxycarbonylamino-4-oxo-hex-2-enoic acid tert-butyl ester (11)
LiCl (162 mg, 3.82 mmol) and TEA (530 µL, 3.80 mmol) were added to a solution of βketophosphonate 12 (1.12 g, 3.80 mmol) in MeCN (30 mL) at 0 °C. Crude tert-butyl
glyoxylate 13 (705 mg, 5.41 mmol) dissolved in MeCN (34 mL) was then added dropwise
followed by stirring for 90 min at 0 °C. The reaction was quenched by addition of citric acid
(10% aq) followed by the addition of Et2O and NaCl (aq, sat.). The phases were separated and
the water phase was extracted twice with Et2O. The combined organic layers were washed
with NaCl (aq, sat.), dried over Na2SO4, filtered and concentrated. The residue was purified
by flash column chromatography (n-heptane:EtOAc 8:1 → 2:1) to give alkene 11 (1.11 g,
total yield of 97% as an isomeric mixture of E:Z 3:1) as a slightly yellow oil. 1H NMR
(CDCl3): δ = 7.10 (d, J = 15.8 Hz, 3H, COCHE), 6.74 (d, J = 15.8 Hz, 3H, CHCOOE), 6.47 (d,
J = 12.1 Hz, 1H, COCHZ), 6.05 (d, J = 12.1 Hz, 1H, CHCOOZ), 5.35-5.21 (m, 4H, NHE and
NHZ), 4.57 (quin, J = 7.0 Hz, 3H, NCHE), 4.47 (quin, J = 7.2 Hz, 1H, NCHZ), 1.50 and 1.47
and 1.44 (3s, 72H, NCOOC(CH3)3 and COOC(CH3)3 for E and Z), 1.39 (d, J = 7.2 Hz, 3H,
CH3 Z), 1.35 (d, J = 7.2 Hz, 9H, CH3 E); 13C NMR (CDCl3) [δ for Z isomer]: δ = 198.4 [202.1]
(CO), 164.3 [164.6] (COO), 155.1 [155.1] (NCOO), 135.0 [137.4] (COCH), 134.5 [128.9]
CHCOO, 82.1 [82.4] (COOC(CH3)3), 80.0 [79.7] (NCOOC(CH3)3), 54.3 [55.2] (NCH), 28.3
[28.3] (C(CH3)3), 27.9 [27.9] (C(CH3)3), 17.6 [17.3] (CH3).
(5S)-5-tert-Butoxycarbonylamino-4-oxo-hexanoic acid tert-butyl ester (18)
Pd/C (15 mg) was added to a solution of alkene 11 (133 mg, 0.447 mmol) in EtOAc (15 mL).
After six vacuum/H2 cycles, the mixture was stirred under an atmospheric pressure of H2 over
night (16 h). The catalyst was removed by filtration through a pad of Celite followed by
concentration and purification by flash column chromatography (n-heptane:EtOAc 5:1 → 4:1)
to give the saturated analogue 18 (109 mg, 81%) as a colorless oil. [α]D +13.3 (c 2.0, CHCl3);
1
H NMR (CDCl3): δ = 5.27 (d, J = 5.9 Hz, 1H, NH), 4.29 (quin, J = 7.1 Hz, 1H, CH), 2.79
(dt, J = 6.8, 18.3 Hz, 1H, COCH2), 2.67 (dt, J = 6.4, 18.3 Hz, 1H, COCH2), 2.53 (dd, J = 6.5,
16.8 Hz, 1H, CH2COO), 2.46 (dd, J = 6.5, 16.8 Hz, 1H, CH2COO), 1.40 and 1.39 (2s, 9H and
9H, NCOOC(CH3)3 and COOC(CH3)3), 1.32 (d, J = 7.1 Hz, 3H, CH3); 13C NMR (CDCl3): δ
= 208.1 (CO), 171.6 (COO), 155.1 (NCOO), 80.6 (COOC(CH3)3), 79.6 (NCOOC(CH3)3),
55.0 (CH), 33.8 (COC H2), 28.9 (CH2COO), 28.2 and 27.9 (NCOOC(C H 3)3 and
COOC(CH3)3), 17.7 (CH3).
37
29
(5S)-5-(9H-Fluoren-9-ylmethoxycarbonylamino)-4-oxo-hexanoic acid (10)
To a solution of 18 (193 mg, 0.640 mmol) in CH2Cl2 (6.5 mL) was TFA (1.3 mL) added at
room temperature. After stirring for 2 h, the solution was concentrated and then redissolved
and concentrated from CHCl3 three times to remove residual TFA. The residue was dissolved
in MeCN:Na2CO3 (10% aq) (1:1, 18 mL) and FmocOSu (227 mg, 0.627 mmol) was added.
The mixture was stirred at room temperature for 17 h followed by concentration to remove the
MeCN. CHCl3 was added and the solution was acidified to pH 2 by addition of HCl (2 M, aq)
at 0 °C. The phases were separated and the water phase was extracted with CHCl3 three times.
The combined organic layers were dried over Na2SO4, filtered and concentrated followed by
purification by flash column chromatography (n-heptane:EtOAc:HOAc 40:10:1 → 25:25:1).
This afforded carboxylic acid 10 (166 mg, 71%) as a white solid. [α]D –14.9 (c 1.0, MeOH);
1
H NMR (CD3OD): δ = 7.82-7.76 (d, J = 7.4 Hz, 2H, Fmoc-arom), 7.69-7.63 (m, 2H, Fmocarom), 7.38 (t, J = 7.4 Hz, 2H, Fmoc-arom), 7.31 (t, J = 7.4 Hz, 2H, Fmoc-arom), 4.57 (brs,
1H, NH), 4.46 (dd, J = 6.8, 10.7 Hz, 1H, Fmoc-CH2), 4.35 (dd, J = 6.7, 10.7 Hz, 1H, FmocCH2), 4.24-4.14 (m, 2H, Fmoc-CH, CH), 2.73 (t, J = 6.2 Hz, 2H, COCH2), 2.56-2.49 (m, 2H,
CH2COO), 1.29 (d, J = 7.2 Hz, 3H, CH3); 13C NMR (CD3OD): δ = 210.7 (CO), 176.2
(COOH), 158.4 (NCOO), 145.3, 145.2, 142.7, 142.6, 128.8, 128.2, 128.1, 126.3, 126.1, 120.9,
67.7 (Fmoc-CH2), 57.0 (Fmoc-CH), 48.3 (CH), 34.2 (COCH2), 28.5 (CH2COO), 16.6 (CH3).
(5S)-4-Oxo-5-((2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropionylamino)-hexanoic acid methyl
ester (21)
Compound 18 (41 mg, 0.133 mmol) was dissolved in CH2Cl2 (8.5 mL) and TFA (1.5 mL)
was added. After stirring for 2 h at room temperature, the solution was concentrated and then
redissolved and concentrated from CHCl3 four times to remove residual TFA. The crude was
dissolved in MeOH (2.0 mL) and trimethylsilyl chloride (59 µL, 0.465 mmol) was slowly
added followed by stirring for 3 h. The solution was concentrated and the crude methyl ester
20 was dissolved in dry CH2Cl2 (2.0 mL). (S)-(+)-α-Methoxy-α-trifluoromethylphenylacetyl
chloride ((S)-MTPA-Cl, 30 µL, 0.160 mmol) was added dropwise followed by addition of
DIPEA (35 µL, 0.199 mmol) at room temperature. The reaction was quenched after 80 min by
the addition of NH4Cl (1 M, aq) and the phases were separated. The water phase was
extracted three times with CH2Cl2 and the combined organic phases were dried over Na2SO4,
filtered and concentrated to give Mosher amide 21. The diastereomeric ratio could not be
determined from the crude due to overlapping signals so it was filtered through a silica
column with care so as to minimize any changes in the isomeric ratio. 1H and 19F NMR
spectroscopy were then used to conclude that the diastereomeric ratio was >100:1.
37
30
(5S/R)-4-Oxo-5-((2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropionylamino)-hexanoic acid
methyl ester (23)
DBU (98 µL, 0.650 mmol) was added to a solution of tert-butyl ester 18 (15 mg, 0.049 mmol)
in THF (1.0 mL) followed by heating at 80 °C for 1800 s by microwave irradiation in a closed
vessel. The solution was concentrated, redissolved in CH2Cl2 and washed twice with citric
acid (10% aq). The combined water phases were extracted twice with CH2Cl2 and the
combined organic layers were then washed with NaCl (aq, sat.), dried over Na2SO4, filtered
and concentrated to give crude racemate 22. [α]D 0.0 (c 1.0, CHCl3). The racemic Mosher
amide 23 was then synthesized from 22 using the same procedure as described for 21.
3-(tert-Butyl-dimethyl-silyloxy)-propan-1-ol (34)
NaH (3.20 g, 133 mmol, 60% in mineral oil) was first washed with pentane three times and
then suspended in THF (250 mL). 1,3-Propanediol (9.5 mL, 131 mmol) was slowly added at
room temperature followed by stirring for 50 min. TBDMSCl (19.8 g, 131 mmol) was added
and vigorous stirring was continued for 60 min. The mixture was poured into Et2O (350 mL)
and was then washed with K2CO3 (10% aq) and NaCl (aq, sat.). The organic phase was dried
over Na2SO4, filtered and concentrated. Purification by flash column chromatography (nheptane:EtOAc 3:1) gave monosilylated alcohol 34 (19.5 g, 78%) as a colorless oil. 1H NMR
(CDCl3): δ = 3.82 (t, J = 5.7 Hz, 2H, SiOCH2), 3.78 (t, J = 5.7 Hz, 2H, HOCH2), 2.68 (s, 1H,
OH), 1.76 (quin, J = 5.6 Hz, 2H, HOCH2CH2), 0.89 (s, 9H, C(CH3)3), 0.06 (s, 6H, SiCH3);
13
C NMR (CDCl3): δ = 62.7 (SiOCH2), 62.1 (HOCH2), 34.2 (HOCH2CH2), 25.8 (C(CH3)3),
18.1 (C(CH3)3), -5.6 (SiCH3).
3-(tert-Butyl-dimethyl-silyloxy)-propionaldehyde (29)
Dess-Martin periodinane (18.5 mL, 8.91 mmol, 15 wt% in CH2Cl2) was added to a solution of
monoprotected alcohol 34 (1.13 g, 5.94 mmol) in CH2Cl2 (25 mL) at room temperature. After
90 min, the reaction was quenched by addition of Na2S2O5 (11.9 g, 62.6 mmol) dissolved in
NaHCO3 (aq, sat.). The phases were separated and the water phase was extracted twice with
CH2Cl2. The combined organic layers were washed with NaHCO3 (aq, sat.), dried over
Na2SO4 and filtered. Concentration and purification by flash column chromatography (nheptane:EtOAc 6:1) gave aldehyde 29 (655 mg, 58%) as a colorless oil of low viscosity. 1H
NMR (CDCl3): δ = 9.80 (dt, J = 0.7, 2.1 Hz, 1H, CHO), 3.98 (dt, J = 0.7, 6.0 Hz, 2H,
SiOCH2), 2.59 (ddt, J = 0.7, 2.1, 6.0 Hz, 2H, CHOCH2), 0.88 (s, 9H, C(CH3)3), 0.06 (s, 6H,
CH3); 13C NMR (CDCl3): δ = 202.0 (CHO), 57.4 (SiOCH2), 46.5 (CHOCH2), 25.8 (C(CH3)3),
18.2 (C(CH3)3), -5.5 (SiCH3).
37
31
2,2,2-Trifluoro-N-((1S)-2-hydroxy-1-methyl-ethyl)-acetamide (32)
To a solution of L-alaninol (2.50 g, 33.3 mmol) in CH2Cl2 (200 mL) was TEA (16.3 mL, 117
mmol) added at room temperature. The solution was cooled to 0 °C and trifluoroacetic
anhydride (6.5 mL, 46.6 mmol) was added during 40 min. After stirring for 6 h at 0 °C, the
solution was concentrated under cold conditions. The crude was redissolved in EtOAc and
then washed with NaHCO3 (aq, sat.). The water phase was extracted twice with EtOAc and
the combined organic layers were then washed with 0.1 M HCl (aq) and NaCl (aq, sat.), dried
over Na2SO4 and filtered. Concentration afforded alcohol 32 (4.43 g, 78%) as a white solid
which was used without further purification. [α]D –16.3 (c 1.0, CHCl3); 1H NMR (CDCl3): δ
= 6.78 (brs, 1H, NH), 4.18-4.07 (m, 1H, CH), 3.75 (dd, J = 3.8, 11.2 Hz, 1H, CH2), 3.62 (dd,
J = 4.9, 11.2 Hz, 1H, CH2), 2.39 (brs, 1H, OH), 1.27 (d, J = 6.9 Hz, 3H, CH3); 13C NMR
(CDCl3): δ = 157.1 (J C-F = 36.9 Hz, CO), 115.8 (JC-F = 288 Hz, CF3), 65.0 (JC-F = 2.1 Hz,
CH2), 47.9 (JC-F = 2.6 Hz, CH), 16.4 (JC-F = 3.0 Hz, CH3).
2,2,2-Trifluoro-N-((1S)-2-iodo-1-methyl-ethyl)-acetamide (35)
PPh3 (7.51 g, 28.6 mmol) and imidazole (2.20 g, 32.3 mmol) were added to a solution of
alcohol 32 (2.04 g, 11.9 mmol) in toluene (140 ml). The solution was slightly heated during
the following addition of I2 (5.80 g, 22.8 mmol). After stirring for 75 min, the solution was
diluted with Et2O (140 ml) and washed with Na2S2O3 (5% aq). The phases were separated and
the aqueous phase was extracted twice with Et2O. The combined organic layers were then
dried over Na2SO4, filtered and concentrated followed by purification by flash column
chromatography (n-heptane:EtOAc 40:1→ 2:1) to afford iodide 35 (2.64 g, 79%) as a white
solid. [α]D –47.2 (c 1.0, CHCl3); 1H NMR (CDCl3): δ = 6.58 (brs, 1H, NH), 3.97-3.87 (m, 1H,
CH), 3.44 (dd, J = 4.9, 10.5 Hz, 1H, CH2), 3.30 (dd, J = 4.5, 10.5 Hz, 1H, CH2), 1.33 (d, J =
6.7 Hz, 3H, CH3); 13C NMR (CDCl3): δ = 156.5 (J = 37.2 Hz, CO), 115.6 (J = 288 Hz, CF3),
45.8 (CH), 20.5 (CH3), 12.0 (CH2).
2,2,2-Trifluoro-N-((1S)-2-bromo-1-methyl-ethyl)-acetamide (36)
A solution of alcohol 32 (101 mg, 0.590 mmol) in MeCN (1.5 mL) was cooled to 0 °C. PPh3
(166 mg, 0.633 mmol) was added followed by dropwise addition of CBr4 (208 mg, 0.627
mmol) dissolved in MeCN (1.5 mL). The solution, which was allowed to slowly reach room
temperature, was stirred for 42 h before CH2Cl2 and H2O were added. The phases were
separated and the water phase was extracted three times with CH2Cl2. The combined organic
layers were washed with NaCl (aq, sat.), dried over Na2SO4, filtered and concentrated. The
residue was purified by flash column chromatography (n-heptane:EtOAc 30:1 → 2:1) to
afford bromide 36 (110 mg, 80%) as a white solid. [α]D –45.7 (c 1.0, CHCl3); 1H NMR
(CDCl3): δ = 6.44 (brs, 1H, NH), 4.38-4.28 (m, 1H, CH), 3.64 (dd, J = 4.2, 10.7 Hz, 1H,
37
32
CH2), 3.49 (dd, J = 3.8, 10.7 Hz, 1H, CH2), 1.37 (d, J = 6.7 Hz, 3H, CH3); 13C NMR (CDCl3):
δ = 156.5 (J = 37.4 Hz, CO), 115.6 (J = 288 Hz, CF3), 45.8 (CH), 37.5 (CH2), 18.7 (CH3).
Triphenyl-[(2S)-2-(2,2,2-trifluoro-acetamido)-propyl]-phosphonium iodide (30)
Iodide 35 (660 mg, 2.35 mmol) was dissolved in toluene (10 mL) and PPh3 (2.48 g, 9.46
mmol) was added. The solution was refluxed for 18 h and then concentrated. The crude was
purified by flash column chromatography (CH2Cl2:MeOH 1:0 → 20:1) to give phosphonium
salt 30 (1.28 g, quantitative yield) as a white amorphous solid. [α]D –17.2 (c 1.0, CHCl3); 1H
NMR (CDCl3): δ = 7.92-7.84 (m, 6H, Ph), 7.83-7.77 (m, 3H, Ph), 7.72-7.64 (m, 6H, Ph), 5.15
(dt, J = 10.8, 15.8 Hz, 1H, CH2), 4.87-4.75 (m, 1H, CH), 3.55 (t, J = 14.8 Hz, 1H, CH2), 1.65
(dd, J = 2.2, 6.9 Hz, 3H, CH3); 13C NMR (CDCl3): δ = 156.5 (J = 38.1 Hz, CO), 135.2,
135.2, 134.0, 133.9, 130.5, 130.3, 117.7, 116.8, 115.2 (J = 288 Hz, CF3), 41.3 (J = 5.05 Hz,
CH), 28.6 (J = 50.2 Hz, CH2), 23.9 (J = 14.5 Hz, CH3).
N-[(1S)-5-(tert-Butyl-dimethyl-silyloxy)-1-methyl-pent-2-enyl]-2,2,2-trifluoro-acetamide (28)
Phosphonium salt 30 (289 mg, 0.532 mmol) was first concentrated from MeOH and dried
under vacuum overnight before being dissolved in THF (4.5 mL) and then cooled to –78 °C.
Titrated n-BuLi (760 µL, 1.06 mmol) was slowly added which resulted in the formation of a
yellow ylide. The solution was allowed to reach room temperature during 20 min, whereupon
it turned black. After cooling to –78 °C again, aldehyde 29 (114 mg, 0.585 mmol) dissolved
in THF (4 mL) was added. The solution was allowed to reach 0 °C during 2 h and then was
Et2O added followed by the addition of 0.1 M HCl saturated with NH4Cl. The phases were
separated and the water phase was extracted with Et2O three times. The combined organic
phases were washed with brine, dried over Na2SO4, filtered and concentration. The crude
alkene displayed an E:Z ratio of 1:1. Purification by flash column chromatography (nheptane:EtOAc 10:1 → 2:1) gave an isomeric mixture of alkene 28 (62 mg, 36%).
Solid-phase peptide synthesis of glycopeptide 37
Glycopeptide 37 incorporating the methylene amine isostere was synthesized as a C-terminal
amide on a Tentagel-S-NH2 resin (Rapp Polymer, Germany) in a manually operated reactor.
Fmoc-2,4-dimethoxy-4´-(carboxymethyloxy)-benzhydrylamine (Rink linker, 4 equiv) was
coupled to the resin, followed by Nα-Fmoc protected amino acids (Bachem, Switzerland and
Neosystem S.A., France, 4 equiv) with the following protective groups on the side chains:
triphenylmethyl (Trt) for glutamine and tert-butyl for glutamic acid. These couplings were
performed using distilled DMF as solvent and the carboxylic acids were activated with
diisopropyl carbodiimide (DIC, 3.9 equiv) and 1-hydroxy-benzotriazole (HOBt, 6 equiv). The
completions of the coupling reactions were monitored using bromophenol blue as indicator.
Fmoc-protected glycosylated hydroxylysine (1.6 equiv) with the Nε-amine protected with a
37
33
carbobenzyloxy (Cbz) group was activated with DIC (1.7 equiv) and 1-hydroxy-7azabenzotriazole (HOAt, 3.4 equiv) and then coupled in DMF for 37 h. The Fmoc-protected
methylene amine isostere 2 was instead coupled by activation with O-(7-azabenzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 2.2 equiv) and 2,4,6-collidine
(3.5 equiv). The completeness of this coupling was monitored by performing a Kaiser test43.
After each coupling cycle, the resin was washed with DMF×5, the Fmoc protective groups
were cleaved by treatment with 20% piperidine in DMF for 10 min (3 min of continuous flow
and 7 min agitating) followed by washing with DMF×5. The N-terminus was finally
acetylated in Ac2O/DMF (2:1) for 2 h but since a Kaiser test indicated that free amines were
still present, the peptide was treated with the same conditions for another 90 min. The resin
was then washed with CH2Cl2×10 and dried on vacuum over night. Subsequent cleavage from
the solid support and deprotection of acid-labile side chain protective groups was
accomplished by treatment with TFA/H2O/thioanisole/ethanedithiol (35:2:2:1, 20 mL) for 3 h
at 40 °C. The resin was filtered off and washed with HOAc followed by concentration. The
crude was then repeatedly concentrated from HOAc and thereafter precipitated from Et2O
followed by lyophilization. Deacetylation was accomplished by treatment with NaOMe (20
mM) for 4 h and the solution was then neutralization by addition of 10% HOAc in MeOH
before concentration. Purification by reversed-phase HPLC (0→100% MeCN (0.1% TFA) in
H2O (0.1% TFA) over 60 min) followed by lyophilization afforded modified glycopeptide 37
(13.8 mg, 26% yield corrected for 70% peptide content) as a white amorphous solid. MS
(MALDI-TOF) calcd. 1054.5 (M+H+), found 1054.7.
37
34
8. Acknowledgements
Jag vill börja med att tacka alla på Organisk kemi, Umeå Universitet, som bidragit till att göra
mitt examensarbete till en väldigt positiv upplevelse.
Några av er vill jag tacka lite extra!
Jan Kihlberg och Anna Linusson för ert engagemang, goda vägledning och för att ni alltid
finns tillgängliga för diskussioner. Tack för att ni gav mig möjligheten att arbeta med detta
projekt som har varit otroligt lärorikt och spännande.
Ett extra stort tack till Tomas Gustafsson för all kunskap du har delat med dig av under den
tid som jag har haft turen att ha dig som handledare. Jag uppskattar verkligen att du har
funnits till hands för att svara på såväl teoretiska som praktiska frågor.
David Blomberg för alla dina tips och goda ideer och för att du har tid att diskutera kemi som
inte riktigt fungerar som man vill. Ett stort tack för din hjälpsamhet och fingerfärdighet med
krånglande HPLC instrument.
Lotta Holm för en utmärkt introduktion till såväl glykopeptidsyntes som upprening med
HPLC och för alla förklaringar till biologirelaterade frågor.
Daniel Wiktelius, Läkemedelskemi, Göteborgs Universitet, för att jag fick ta del av din
syntesväg för E-alkenisosterer och för alla tips angående de olika reaktionerna.
Mattias Hedenström för all hjälp med att köra NMR på glykopeptiderna.
37
35
9. References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Engelhard, V. H. How cells process antigens. Sci. Am. 1994, Aug., 44-51.
Astorga, G. P.; Williams, R. C. Altered reactivity in mixed lymphocyte culture of lymphocytes from
patients with rheumatoid arthritis. Arthritis Rheum. 1969, 12, 547-554.
Stastny, P. Mixed lymphocyte cultures in rheumatoid arthritis. J. Clin. Invest. 1976, 57, 1148-1157.
Trentham, D. E.; Townes, A. S.; Kang, A. H. Autoimmunity to type II collagen an experimental
model of arthritis. J. Exp. Med. 1977, 146, 857-868.
Courtenay, J. S.; Dallman, M. J.; Dayan, A. D.; Martin, A.; Mosedale, B. Immunisation against
heterologous type II collagen induces arthritis in mice. Nature 1980, 283, 666-8.
Brunsberg, U.; Gustafsson, K.; Jansson, L.; Michaëlsson, E.; Ährlund-Richter, L.; Pettersson, S.;
Mattson, R.; Holmdahl, R. Expression of a transgenic class II Ab gene confers susceptibility to
collagen-induced arthritis. Eur. J. Immunol. 1994, 24, 1698-1702.
Michaëlsson, E.; Andersson, M.; Engström, Å.; Holmdahl, R. Identification of an immunodominant
type-II collagen peptide recognized by T cells in H-2q mice: self tolerance at the level of determinant
selection. Eur. J. Immunol. 1992, 22, 1819-1825.
Corthay, A.; Bäcklund, J.; Broddefalk, J.; Michaëlsson, E.; Goldschmidt, T. J.; Kihlberg, J.;
Holmdahl, R. Epitope glycosylation plays a critical role for T cell recognition of type II collagen in
collagen-induced arthritis. Eur. J. Immunol. 1998, 28, 2580-2590.
Holm, L.; Kjellén, P.; Holmdahl, R.; Kihlberg, J. Identification of the minimal glycopeptide core
recognized by T cells in a model for rheumatoid arthritis. Bioorg. Med. Chem. 2005, 13, 473-482.
Rosloniec, E. F.; Whittington, K. B.; Brand, D. D.; Myers, L. K.; Stuart, J. M. Identification of MHC
class II and TCR binding residues in the type II collagen immunodominant determinant mediating
collagen-induced arthritis. Cell. Immunol. 1996, 172, 21-28.
Kjellén, P.; Brunsberg, U.; Broddefalk, J.; Hansen, B.; Vestberg, M.; Ivarsson, I.; Engström, A.;
Svejgaard, A.; Kihlberg, J.; Fugger, L.; Holmdahl, R. The structural basis of MHC control of
collagen-induced arthritis; binding of the immunodominant type II collagen 256-270 glycopeptide to
H-2Aq and H-2Ap molecules. Eur. J. Immunol. 1998, 28, 755-767.
Holm, B.; Bäcklund, J.; Recio, M. A. F.; Holmdahl, R.; Kihlberg, J. Glycopeptide specificity of
helper T cells obtained in mouse models for rheumatoid arthritis. ChemBioChem 2002, 3, 12091222.
Hedenström, M.; Holm, L.; Yuan, Z.; Emtenäs, H.; Kihlberg, J. Stereoselective synthesis of
ψ[CH2O] pseudodipeptides and conformational analysis of a Pheψ[CH2O]Ala containing analogue
of the drug desmopressin. Bioorg. Med. Chem. Lett. 2002, 12, 841-844.
Holm, L.; Bockermann, R.; Holmdahl, R.; Kihlberg, J. Peptide requirements for stimulation of
glycopeptide specific T cells obtained in a mouse model for rheumatoid arthritis. Manuscript 2005.
Bäcklund, J.; Treschow, A.; Bockermann, R.; Holm, B.; Holm, L.; Issazadeh-Navikas, S.; Kihlberg,
J.; Holmdahl, R. Glycosylation of type II collagen is of major importance for T cell tolerance and
pathology in collagen-induced arthritis. Eur. J. Immunol. 2002, 32, 3776-3784.
Holmdahl, R.; Andersson, E. C.; Andersen, C. B.; Svejgaard, A.; Fugger, L. Transgenic mouse
models of rheumatoid arthritis. Immunol. Rev. 1999, 169, 161-173.
McFarland, B. J.; Beeson, C. Binding interactions between peptides and proteins of the class II major
histacompatibility complex. Med. Res. Rev. 2002, 22, 168-203.
Stern, L. J.; Wiley, D. C. Antigenic peptide binding by class I and class II histocompatibility
proteins. Structure 1994, 2, 245-251.
Loughlin, W. A.; Tyndall, J. D. A.; Glenn, M. P.; Fairlie, D. P. Beta-strand mimetics. Chem. Rev.
2004, 104, 6085-6117.
Venkatesan, N.; Kim, B. H. Synthesis and enzyme inhibitory activities of novel peptide isosteres.
Curr. Med. Chem. 2002, 9, 2243-2270.
37
36
21. Bolin, D. R.; Swain, A. L.; Sarabu, R.; Berthel, S. J.; Gillespie, P.; Huby, N. J. S.; Makofske, R.;
Orzechowski, L.; Perrotta, A.; Toth, K.; Cooper, J. P.; Jiang, N.; Falcioni, F.; Campbell, R.; Cox, D.;
Gaizband, D.; Belunis, C. J.; Vidovic, D.; Ito, K.; Crowther, R,; Kammlott, U.; Zhang, X.; Palermo,
R.; Weber, D.; Guenot, J.; Nagy, Z.; Olson, G. L. Peptide and peptide mimetic inhibitors of antigen
presentation by HLA-DR class II MHC molecules. Design, structure-activity relationships, and Xray crystal structures. J. Med. Chem. 2000, 43, 2135-2148.
22. Mézière, C.; Viguier, M.; Dumortier, H.; Lo-Man, R.; Leclerc, C.; Guillet, J.-G.; Briand, J.-P.;
Muller, S. In vivo T helper cell response to retro-inverso peptidomimetics. J. Immunol. 1997, 159,
3230-3237.
23. Ettouati, L.; Salvi, J. P.; Trescol-Biémont, M. C.; Walchshofer, N.; Gerlier, D.; Rabourdin-Combe,
C.; Paris, J. Substitution of peptide bond 53-54 of HEL(52-61) with an ethylene bond rather than
reduced peptide bond is tolerated by an MHC-II restricted T cell. Pep. Res. 1996, 9, 248-253.
24. The comparative modeling of Aq was performed on the course Drug Design and Combinatorial
Chemistry given at the department of Organic Chemistry, Umeå University, and hence was not a part
of this master thesis.
25. Gold version 2.2 for Linux supplied by The Cambridge Crystallographic Data Centre (CCDC),
Cambridge, UK, http://www.ccdc.cam.ac.uk/products/life_sciences/gold).
26. Aherton, E.; Logan, C. J.; Sheppard, R. C. Procedures for solid-phase synthesis using Nαfluorenylmethoxycarbonylamino acids on polyamide supports. Synthesis of substance P and of acyl
carrier protein 65-74 decapeptide. J. Chem. Soc. Perkin Trans. 1, 1981, 538-546.
27. Kokotos, G. A convenient one-pot conversion of N-protected amino-acids and peptides into alcohols.
Synthesis-Stuttgart, 1990, 4, 299-301.
28. Myers, A. G.; Zhong, B.; Movassaghi, M.; Kung, D. W.; Lanman, B. A.; Kwon, S. Synthesis of
highly epimerizable N-protected α-amino aldehydes of high enantiomeric excess. Tet. Lett. 2000, 41,
1359-1362.
29. Våbenø, J.; Brisander, M.; Lejon, T.; Luthman, K. Diastereoselective reduction of a chiral N-bocprotected δ-amino-α,β-unsaturated γ-keto ester Phe-Gly dipeptidomimetic. J. Org. Chem. 2002, 67,
9186-9191.
30. Schuda, P. F.; Ebner, C. B.; Potlock, S. J. The synthesis of 4-oxo-2-pentenoic esters by Wittig
reaction using α-oxoesters. Synthesis-Stuttgart 1987, 12, 1055-1057.
31. Déziel, R.; Plante, R.; Caron, V.; Grenier, L.; Llinas-Brunet, M.; Duceppe, J.-S.; Malenfant, E.;
Moss, N. A practical and diastereoselective synthesis of ketomethylene dipeptide isosteres of the
type AAψ[COCH2]Asp. J. Org. Chem. 1996, 61, 2901-2903.
32. Dale, J. A.; Dull, D. L.; Mosher, H. S. α-Methoxy-α-trifluoromethylphenylacetic acid, a versatile
reagent for the determination of enantiomeric composition of alcohols and amines. J. Org. Chem.
1969, 34, 2543-2549.
33. Wiktelius, D.; Luthman, K. Unpublished results.
34. McDougal, P. G.; Rico, J. G.; Oh, Y.-I.; Condon, B. D. A convenient procedure for the
monosilylation of symmetric 1,n-diols. J. Org. Chem. 1986, 51, 3388-3390.
35. Wellner, E.; Gustafsson, T.; Bäcklund, J.; Holmdahl, R.; Kihlberg, J.; Synthesis of a C-glycoside
analogue of β-D-galactosyl hydroxynorvaline and its use in immunological studies. ChemBioChem
2000, 1, 272-280.
36. Maryanoff, B. E.; Reitz, A. B. The Wittig olefination reaction and modifications involving
phosphoryl-stabilized carbanions. Stereochemistry, mechanism, and selected synthetic aspects.
Chem. Rev. 1989, 89, 863-927.
37. Zhu, Y.; Rudensky, A. Y.; Teyton, A. L.; Wilson, I. A. Crystal structure of MHC class II I-Ab in
complex with a human clip peptide: Prediction of an I-Ab peptide-binding motif. J. Mol. Biol. 2003,
326, 1157-1174.
38. Hooft, R. W. W.; Vriend, G.; Sander, C.; Abola, E. E. Errors in protein structures. Nature 1996, 381,
272-272.
37
39. Word, J. M.; Lovell, S. C.; Richardson, J. S.; Richardson, D. C. Asparagine and glutamine: Using
hydrogen atom contacts in the choice of side-chain amide orientation. J. Mol. Biol. 1999, 285, 17351747.
40. MOE version 2004.03 for Linux, Chemical Computing Group Inc., S. S., West Suite, 910 Montreal,
Canada H3A 2R7, 2004.
41. www2.ccc.uni-erlangen.de/software/corina/free_struct.html.
42. Andersson, D.; Sjövik Johansson, E.; Bylesjö, M.; Lindström, A.; Raubacher, F.; Linusson, A.
Manuscript in preparation.
43. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test for detection of free terminal
amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 1970, 34, 595-598.
37
38