Synthesis and evaluation of a (3R,6S,9S)-2-oxo-1-azabicyclo[4.3.0]nonane scaffold as a mimic of Xaa-trans-Pro in poly-l-proline type II helix conformation

Article abstract of DOI:10.1039/c5ob00180c

We describe the development of a small-molecule mimic of Xaa-trans-Pro dipeptide in poly-l-proline type II helix conformation, based upon a (3R,6S,9S)-2-oxo-1-azabicyclo[4.3.0]nonane core structure. Stereoselective synthesis of the mimic from l-pyroglutamic acid is achieved in twelve linear steps and 9.9% yield. Configurational and conformational analyses are conducted using a combination of ¹H NMR spectroscopy, X-ray crystallography and circular dichroism spectroscopy; and evaluation of the mimic as a promising surrogate dipeptide, in a protein-protein interaction between the SH3 domain of human Fyn kinase (Fyn SH3) and peptidomimetics of its biological ligand, are conducted by ¹H-¹⁵N HSQC NMR titration experiments.

Full text of DOI:10.1039/c5ob00180c

Organic &
Biomolecular Chemistry
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PAPER
Synthesis and evaluation of a (3R,6S,9S)-2-oxo-
-azabicyclo[4.3.0]nonane scaffold as a mimic
of Xaa-trans-Pro in poly-L-proline type II helix
conformation†
1
Cite this: Org. Biomol. Chem., 2015,
3, 4562
1
a
b
c
a
Boris Aillard, Jeremy D. Kilburn, Jeremy P. Blaydes, Graham J. Tizzard,
d
d
a
Stuart Findlow, Jörn M. Werner and Sally Bloodworth*
We describe the development of a small-molecule mimic of Xaa-trans-Pro dipeptide in poly-L-proline
type II helix conformation, based upon a (3R,6S,9S)-2-oxo-1-azabicyclo[4.3.0]nonane core structure.
Stereoselective synthesis of the mimic from L-pyroglutamic acid is achieved in twelve linear steps and
1
9
.9% yield. Configurational and conformational analyses are conducted using a combination of H NMR
Received 29th January 2015,
Accepted 10th March 2015
spectroscopy, X-ray crystallography and circular dichroism spectroscopy; and evaluation of the mimic as
a promising surrogate dipeptide, in a protein–protein interaction between the SH3 domain of human Fyn
DOI: 10.1039/c5ob00180c
1
15
kinase (Fyn SH3) and peptidomimetics of its biological ligand, are conducted by H- N HSQC NMR titra-
tion experiments.
www.rsc.org/obc
structural motifs for possible therapeutic intervention in
protein–protein interactions.
Introduction
The poly-L-proline type II (PPII) helix is an element of second-
To date, only Kühne and Schmalz have combined rational
ary structure characterised by a left-handed helix of three resi- design, synthesis and conformational analysis of a small mole-
dues per turn, a helical pitch of 9.3 Å, and torsional angles cule PPII-mimetic scaffold with measurement of the binding
with distribution maxima of ϕ = −75° and ψ = 145°. The major of a peptide ligand incorporating the mimetic in a protein–
role played by the PPII helix in protein structure and function protein interaction characterised by PPII conformation in that
1
3m
has been widely reviewed and, in particular, the intercession ligand. We sought to similarly apply a systematic approach
of PPII-helical peptides in protein–protein interactions to the development of a new conformational mimetic based
2
between proline-rich regions and their recognition domains
upon a (3R,6S,9S)-2-oxo-1-azabicyclo[4.3.0]nonane core struc-
has highlighted the potential of small-molecule peptidomi- ture 1. The candidate structure was identified as a potential
3
metics of PPII conformation as tools for the study of the inter- Xaa-trans-Pro dipeptide surrogate via screening of a series of
action of the PPII helix with its recognition domains, or as azabicyclo[X.Y.0]alkane/-ene candidates for optimal constraint
in their energy-minimised conformation, of dihedral angles
corresponding to ω, ϕ and ψ to those which define PPII confor-
mation (Fig. 1).
a
Chemistry, Faculty of Natural and Environmental Sciences,
University of Southampton, Southampton, SO17 1BJ, UK.
E-mail: S.Bloodworth@soton.ac.uk
The DFT-optimised structure of (3R,6S,9S)-1 predicts di-
hedral angles of ω = 174.6°, ϕ = −68.5° and ψ = 144.3°; each
well-centred within known permitted deviation for these PPII
b
School of Biological and Chemical Sciences, Queen Mary, University of London,
Mile End Road, London, E1 4NS, UK
c
Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton,
1b
3
conformation descriptors. Diagnostic J coupling constants
SO16 6YD, UK
3
3
d
for the ring-junction proton H6, of J
H6–H5
≈ J
H6–H7
= 2.8–4.3
Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ,
3
3
4
UK
Hz and JH6–H5′ ≈ JH6–H7′ = 9.8–10.2 Hz are predicted if the
conformation of (3R,6S,9S)-1 in solution is to prove consistent
with the calculated structure. Dipeptide surrogates based
†
1
Electronic supplementary information (ESI) available: Experimental details,
13
H and C NMR spectra for preparation and characterisation of all new com-
pounds. Fyn SH3 expression and purification, NMR assignment of Fyn SH3 and
binding data for titration against 12–15. Structural data. CCDC 1014671. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c5ob00180c
5
upon the diastereomeric (3S,6S,9S)-2-oxo-1-azabicyclo[4.3.0]-
nonane structure have been widely explored, and recently
6
reviewed, as mimics of β-turn conformation.
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Fig. 1 (a) Xaa-trans-Pro mimic candidate (3R,6S,9S)-1. (b) Optimised
7
geometry for (3R,6S,9S)-1 was obtained by DFT using B3LYP/6-31G(d, p).
Partial atom labelling is shown for clarity. Calculated dihedral angles
correspond to ω, ϕ and ψ of Xaa-trans-Pro in PPII conformation as
indicated in square brackets.
Scheme 1 Synthesis of N-Boc(3R,6S,9S)-1-OMe. Reagents and con-
ditions: (a) MeOH, Dowex®, Δ, 16 h, 96%; (b) Boc
°C to room temp., 82%; (c) L-selectride, THF, −78 °C then MeOH,
p-TsOH (0.1 equiv.); 52% over two steps; (d) BF ·Et O, Me SiCH CHvCH
78 °C, 77%; (e) TFA, CH Cl , 55%; (f) NaH, benzyl chloroformate,
8%; (g) O , −78 °C then PPh , 96%; (h) (MeO) P(O)CH(NHCbz)CO Me,
BuOK, −78 °C to room temp., 5 h, 79%; (i) Rh(I)COD-(R,R)-Et-
DuPHOS, H (100 psi), 24 h, 90%; ( j) Boc O, DMAP, CH CN, room temp.,
7%; (k) H , Pd/C, MeOH, 16 h, 98%.
2 3
O, DMAP, CH CN,
0
Results and discussion
3
2
3
2
2
,
−
8
tert
2
2
Preparation of 1 was carried out by consolidation of estab-
3
3
2
2
lished procedures for synthesis of a 5-substituted L-proline
8
methyl ester derivative 6, as precursor to the (3R,6S,9S)-2-
2
2
3
oxo-1-azabicyclo[4.3.0]nonane core motif via hydrogenolysis
9
2
9
and lactam cyclisation (Scheme 1). Hence, conversion of
enantiopure L-pyroglutamic acid (2) to methyl (2S)-1-(tert-
butoxycarbonyl)-pyroglutamate, followed by reduction with
L-selectride and subsequent conversion to the hemiaminal Whilst these expected nOes were indeed present in the
ether, allowed the multigram preparation of 3. Treatment of spectra of N-Boc(3S,6S,9S)-1-OMe, overlap of the ring-junction
the N-acyliminium ion derived from 3 with allyltrimethylsilane proton H6 with the methyl group resonance occurred in the
8
c
1
at low temperature gave the products of C5-allylation as an
H spectrum of N-Boc(3R,6S,9S)-1-OMe such that irradiation
inseparable 3 : 1 cis–trans mixture. N-deprotection then of H6 was impossible. We therefore confirmed the apparent
1
0
resulted in clean separation of the (2S,5R) cis-isomer 4. Rela- lack of nOe between H3 and H6 in the desired 3R-epimer
tive stereochemistry in 4 was confirmed by nOe correlation by direct hydrogenolysis/δ-lactamisation of 6, to provide
1
between protons at the 2- and 5-position stereogenic centres. N-H(3R,6S,9S)-1-OMe in which there is no overlap in the H NMR
Introduction of benzyloxy carbamate N-protection and ozono- spectrum. No nOe correlation of H3 with either H6 or H9 is
1
1
lysis followed by (Z)-selective olefination, then afforded 5. observed for N-H(3R,6S,9S)-1-OMe (Fig. 2). The relative con-
Rhodium-catalysed asymmetric hydrogenation of the mixture figuration of the 2-oxo-1-azabicyclo[4.3.0]nonane product was
1
2
of isomers 5, using Rh(I)COD-(R,R)-Et-DuPHOS, furnished a further determined by single crystal diffraction† and
single diastereomer 6 (>90% de) in which the expected conver- (3R,6S,9S) absolute configuration inferred from that of 2.
3
gence of the geometrically impure substrate to R-configuration
J coupling constants for resonances arising from H3, H6 and
1
at the newly created stereogenic centre in 6 is verified by extra- H9 in the H NMR spectrum of N-H(3R,6S,9S)-1-OMe, and di-
polation of 3R-configuration in 1 (vide infra). Finally, treatment hedral angles ω, ϕ and ψ determined from the solid-phase
of 6 with Boc O, followed by hydrogenolysis, led to Xaa-trans- structure (Fig. 3) are consistent with those expected for the
2
Pro dipeptide mimic 1, the product of intramolecular δ-lacta- intended (PPII-mimetic) conformation predicted by DFT.
misation, with suitable N-Boc and C-methyl ester terminus Moreover, the ϕ and ψ pair fall within the boundary of a
protection for incorporation into standard peptide coupling region of the Ramachandran plot defining PPII conformation
protocols.
The relative configuration of 1 was confirmed by a combi- of protein structure.
nation of nOe experiments and crystallographic analysis. We
Conformational studies upon short-chain oligomers of
sought to distinguish N-Boc(3R,6S,9S)-1-OMe from its 3S- (3R,6S,9S)-1 were next undertaken. The dimer (9), trimer (10)
by the clustering of PPII helices determined through surveys
1
b
1
3
epimer by the respective absence (3R-isomer) and presence and tetramer (11) represent 4-, 6- and 8-residue peptide
3S-isomer) of an nOe correlation of H3 with both H6 and H9. mimics respectively, and were prepared using standard
(
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torsional angles of an extended PPII conformation, whilst ψ′
does not (Fig. 4).
Circular dichroism (CD) analysis of 9–11 was undertaken in
methanol and in water and, in each case, gave spectra which
exhibited a strong negative maximum at 208 nm in methanol
and at 202 nm in water (π→π*), and a weak positive maximum
i i
at 226–8 nm in methanol and 224 nm in water (Oi−1⋯C ′vO ,
1
4
n→π*) which we attribute to PPII conformation within those
sections of the peptidomimetic backbone which are ordered as
a result of the conformationally locked subset of ω, ϕ and ψ
(
Fig. 5). Interestingly, monomer N-Boc(3R,6S,9S)-1-OMe does
not display clear PPII conformational order, and a decrease in
1
5
the relative band strength ρ(θmax/θmin
)
indicative of increas-
ing conformational stability is then observed with increasing
chain length 9→11 in both solvents, arising predominantly
from an increase in intensity of the π→π* CD signal. Mean
molar ellipticity was found to be independent of sample con-
1
6
centration, suggesting that there is no intermolecular stabil-
isation of an induced PPII conformation by backbone
solvation, which is described to stabilise the PPII helix in pure
1
7
1
polypeptides. Rather, a favourable n→π* interaction may
3
Fig. 2 H NMR (400 MHz, CDCl ) features, diagnostic of conformation
and relative configuration in 2-oxo-1-azabicyclo[4.3.0]nonane mimetics stabilise trans-conformation in the exocyclic amide bond(s) of
of Xaa-trans-Pro.
9
–11 leading to an overall cooperative stabilisation of helical
conformation, related to chain length, via an induced dihedral
angle ϕ′ close to that describing the PPII helix (and consistent
1
4b,18
with that observed in the solid state, Fig. 4).
Interpretation of CD spectra in order to identify confor-
1
9
mational order is not necessarily straightforward however,
and it is important to emphasise that there is here insufficient
evidence to conclude that an induced helical conformation in
the exocyclic regions of 9–11 exists in solution. Indeed, in the
1
3
H NMR spectra of 9–11, J
coupling constants between 5.6
HNα
Hz (9, 400 MHz, CDCl
correspond to ϕ′ = −71° and −87° respectively, which are dis-
tributed around the median values of J
3 3
) and 7.6 Hz (10, 11, 400 MHz, CD CN)
2
0
3
= 6.5 Hz, ϕ = −71°
HNα
2
1
characteristic of PPII conformation; but an αN(i, i + 1) nOe
correlation between H9 and NH10′ (which would be indicative
that ψ′ occupies the area of Ramachandran space diagnostic of
2
1b
PPII helicity) is not observed for 9, 10 or 11.
We next sought to explore the potential of the (3R,6S,9S)-2-
oxo-1-azabicyclo[4.3.0]nonane scaffold as an Xaa-trans-Pro
mimic in peptidomimetics. Well-documented structural and
2
e
interaction studies exist for the proline-rich, 14 amino acid
peptide PPRPLPVAPGSSKT which adopts a PPII conformation
Fig. 3 (a) X-Ray crystal structure of (3R,6S,9S)-1-OMe·HCl and
measured dihedral angles ω, ϕ and ψ. Solvent excluded, partial atom upon complex formation with its biological target. The peptide
labelling for clarity; (b) Overlay of (3R,6S,9S)-1-OMe·HCl (orange) and
DFT-optimised minimum energy conformer of (3R,6S,9S)-1 (cyan).
corresponds to residues 91-104 of the p85α regulatory subunit
of human phosphoinositide 3-kinase and is the binding motif
involved in the protein’s interaction with the SH3 domain of
2
2
human Fyn kinase (Fyn SH3). In the complex, key intermole-
cular nOe contacts are observed for Arg3, Leu5, Pro6, Ala8
procedures of solution-phase peptide coupling (Scheme 2). and Pro9 of PPRPLPVAPGSSKT, and we therefore selected
We define a second set of dihedral angles (′) in 9–11, which the shortened peptide RPLPVAPG as a surrogate to test the
correspond to ω′, ϕ′ and ψ′ of the peptidomimetic, but are exo- effects of incorporation of the Xaa-trans-Pro dipeptide mimic
cyclic to those of the core (3R,6S,9S)-2-oxo-1-azabicyclo[4.3.0]- on the modified peptide’s interaction with Fyn SH3 using
nonane motif and thus unconstrained. Indeed, in the solid- NMR. The C-terminus glycine residue is retained to aid
phase conformation of 9 only ω′ and ϕ′ approximate the solubility.
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Scheme 2 Solution-phase synthesis of oligomers 9–11. Reagents and conditions: (a) CF
3
CO
2
H (20% v/v in CH
2
Cl
2
), room temp., 16 h; (b) 1 M LiOH,
i
THF, room temp., 1 h; (c) EDC, HOBt, Pr
2
EtN, DMF, 0 °C to room temp.
2
N-Ac-RPLPVAPG-NH (12) was confirmed as a suitable surro-
gate for the protein–protein interaction between p85α and
Fyn SH3 since chemical shift perturbation (ΔδHN
0
.2–0.8 ppm) of backbone NH resonances corresponding to
involvement of Trp119, Asn136, Tyr137 and Tyr91 (of Fyn
SH3) in the binding event were observed. Binding curves†
were determined for all resonances in the region ΔδHN
>
0
.2 ppm by plotting fractional shift (Δδ/Δδmax) against [12]
and an average dissociation constant of K = 41 μM was calcu-
d
lated, in good agreement with that reported for binding of
the 14mer homologue PPRPLPVAPGSSKT (K = 50 μM) by
d
2
e
Morton et al. Location of the binding site in the expected
region of Fyn SH3 is depicted in Fig. 7(a). Pleasingly, substi-
tution of the AlaPro dipeptide sequence by the mimetic
Fig. 4 X-Ray crystal structure of
dihedral angles ω’, ϕ’ and ψ’. Solvent excluded, partial numbering
for clarity.
9 and measured unconstrained
(
3R,6S,9S)-1, did not markedly affect the binding orientation
of resulting peptidomimetic ligand 13 [Fig. 7(b)], although
weaker association (K = 300 μM averaged across nine reson-
d
ances with ΔδHN > 0.17 ppm) took place, presumably as a
result of the loss of key contacts with Trp119 and Tyr91 which
have been characterised for the alanine residue of
PPRPLPVAPGSSKT. It is worth noting that key contacts
between the leucine residue of ligand 13, and SH3 residues
Gly117, Asp118 and Tyr137 appear to be maintained.
The leucine side-chain is absent from ligands 14 and 15,
following peptidomimetic replacement of LeuPro with the
backbone mimic (3R,6S,9S)-1, a defined interaction between
ligands 14 or 15 and Fyn SH3 was not measurable upon
NMR titration. Small chemical shift perturbations, dispa-
rately located around the protein, were seen in each case
Mimetic substitution of the dipeptide regions LeuPro and/
or AlaPro of RPLPVAPG defines 3 potential PPII mimics 13–15.
N-Ac-RPLPVAPG-NH
3–15 were prepared using conventional Fmoc solid-phase syn-
thesis, supported on Rink amide resin (Scheme 3).
The expected binding model for Fyn SH3-(N-Ac-
RPLPVAPG-NH ) complex was extrapolated from that reported
2
(12) and peptidomimetic analogues
1
a
2
2
e
for Fyn SH3-(PPRPLPVAPGSSKT) in which intermolecular
nOe crosspeaks in the solution-phase NMR structure indicate
that residues Trp119, Asn136, Tyr137 and Tyr91 of the Fyn SH3
domain are involved in interaction with the peptide ligand
[
Fig. 7(b) and (c)] and suggest only weak and non-specific
association.
(
Fig. 6).
We obtained NMR assignments of backbone resonances
1
3
15
for C/ N Fyn SH3 using standard triple resonance measure-
1
15
ment and sequential assignment techniques, and H- N
HSQC spectra were then recorded upon titration of each
Conclusions
ligand (12–15) in various ratios against the protein in pH 6 In summary, we have described the rational design, stereo-
potassium phosphate buffer solution with co-solvents 5% v/v selective synthesis and conformational analysis of a (3R,6S,9S)-
D O and 10% v/v DMSO-d to aid ligand solubility. Binding of 2-oxo-1-azabicyclo[4.3.0]nonane scaffold, (3R,6S,9S)-1, as a
2 6
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Scheme 3 Solid-phase synthesis of N-Ac-RPLPVAPG-NH
analogues 13–15. Reagents and conditions: (a) 1 M LiOH, THF, room
temp., 1 h; (b) CF CO H (20% v/v in CH Cl ), room temp.; (c) Fmoc-OSu,
Na CO (sat. aq. solution), dioxane, room temp.; (d) piperidine (20% v/v
in DMF), 20–30 min; (e) FmocAA-OH (AA-OH = Gly-OH, Pro-OH, Ala-
2
(12) and
3
2
2
2
2
3
i
OH, Val-OH, Leu-OH or (3R,6S,9S)-1-OH), HOBt, DIC, Pr
2
NEt, DMF,
NEt, DMF, room
O (50% v/v in pyridine), room temp., 45 min; (g)
SiH–H O (96 : 2 : 2 v/v), room temp., 2 h.
i
room temp., 5 h, or FmocArg(Pbf)-OH, HBTU, Pr
temp., 5 h; (f) Ac
2
2
i
CF
3
CO
2
H– Pr
3
2
Fig. 5 Circular dichroism spectra in (a) MeOH and (b) water. Spectra
were recorded at 23 °C and concentrations of N-Boc(3R,6S,9S)-1-OMe =
0
.5 mM, 9 = 0.5 mM, 10 = 0.25 mM, 11 = 0.125 mM; and are normal-
ised for concentration and to give θ = mean molar ellipticity per amide
bond. Relative band strengths ρ(θmax/θmin) are as follows; 9: ρMeOH = 0.33
and ρwater = 0.91; 10: ρMeOH = 0.22 and ρwater = 0.31; 11: ρMeOH = 0.19
and ρwater = 0.22.
2
Fig. 6 Simulated binding of N-Ac-RPLPVAPG-NH (12) in PPII confor-
2e
mation by Fyn SH3 domain, extrapolated from Morton et al. Residues
displaying reported nOe crosspeaks with PPRPLPVAPGSSKT are shown
in green: Trp119, Asn136, Tyr137 and Tyr91 from left→right.
promising small-molecule dipeptide mimic in poly-L-proline
type II helix conformation. Peptidomimetic ligands featuring
Xaa-trans-Pro sequence replacement by the mimic demonstrate
a general loss of affinity and selectivity for targeted binding to
Fyn SH3 with increasing peptidomimetic content, corresponding ising core ‘backbone’ conformational mimetic of regions of
to loss of side-chain functionality totaling four of the five resi- trans-Pro-, Ala- and Gly-rich primary sequence adopting PPII
dues involved in specific components of the protein–protein secondary structure. Our continuing work in this area, towards
interaction between Fyn SH3 and the ‘parent’ peptide ligand. useful pseudo-peptide compounds, will involve the develop-
However, in maintaining important features of the ligand- ment of a second generation of (3R,6S,9S)-2-oxo-1-azabicyclo-
protein complex 13-Fyn SH3 following transposition of Ala- [4.3.0]nonane scaffolds, suitably functionalised at C3 with sub-
trans-Pro by (3R,6S,9S)-1 we suggest that (3R,6S,9S)-1 is a prom- stituents for mimicry of the L-amino acid side-chain.
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studies were carried out on samples containing 5% DMSO to
aid mimetic solubility. No change was observed in the K of the
d
native peptide (12) for Fyn SH3 under these conditions.
The peptide binding site was obtained by recording a series
1
15
of nine 2D H- N-HSQC spectra at peptide/protein ratios of
0
2
: 1, 0.25 : 1, 0.5 : 1, 0.75 : 1, 1 : 1, 2 : 1, 4 : 1, 8 : 1, 12 : 1 and
0 : 1 with the protein at 0.1 mM. K values were obtained for
d
residues in the fast-exchange regime by fitting the fractional
shift:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ðKd þ ½Lꢀ þ ½PꢀÞ ꢁ ðKd þ ½Lꢀ þ ½PꢀÞ ꢁ 4½Pꢀ½Lꢀ
Δδ=Δδmax ¼
2
½Pꢀ
where Δδ/Δδmax is the fractional shift and [L] and [P] are the
ligand (peptide or mimetic) and protein (Fyn SH3) concen-
trations respectively. The backbone chemical shift pertur-
1
bation upon peptide addition was calculated by weighting HN
1
5
and N chemical shift differences as follows:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ
1
2
2
ΔδH;N ¼
ðΔδHÞ þ ðΔδNÞ
6
where Δδ
H
and Δδ
N chemical shifts, respectively, of the fully saturated and free
protein.
N
are the differences between the HN and
1
5
2
5
All NMR data were processed with NMRPipe, using mild
resolution enhancement and linear prediction in the hetero-
nuclear dimension, before being subjected to Fourier trans-
1
15
Fig. 7 Histograms of H- N chemical shift change for residues of Fyn
SH3 upon binding of (a) 12 (b) 13 (c) 14 and (d) 15. Residues with no formation. The resulting spectra were analysed with CcpNmr
2
6
amide proton (proline) are not shown. In each case, residues showing Analysis software.
ΔδHN > 0.2 ppm for binding of 12, ΔδHN > 0.17 ppm for binding of 13,
ΔδHN > 0.1 ppm for binding of 14, and ΔδHN > 0.075 ppm for binding of
Circular dichroism spectroscopy
1
5, are highlighted in green on the known X-ray structure of free human
2
3
Fyn SH3.
CD measurements were performed at 25 °C with a Jasco J720
spectrophotometer at a spectral bandwidth of 2 nm with a
time constant of 2 s (scan speed 100 nm min− ) and a step
resolution of 1 nm. Spectra were recorded as an average of 16
scans. For each spectrum, the background was subtracted
prior to smoothing and processing. Machine data units of
millidegrees ellipticity were normalised as a function of the
concentration of solutions and the number of amide bonds by
conversion to mean residue molar ellipticity (θ) using the fol-
lowing equation (where n is the number of peptide bonds and
Ellipticity is the raw data from the instrument).
1
Methods
Protein preparation
The gene for the Fyn SH3 domain (residues 73–145) encoded
with an N-terminal GST tag in the pGEX-2 T plasmid was a
kind gift from Xavier Morelli and Stéphane Betzi (Cancer
Research Center of Marseille). The plasmid was transformed
into E. coli cell line BL21-CodonPlus(DE3)-RIPL (Agilent
Technologies) and expressed and purified according to
2
e
6
EllipticityðmdegÞ ꢂ 10
methods adapted from Campbell et al.†
θðdeg cm dmolꢁ1Þ ¼
2
PathlengthðmmÞ½CꢀðμMÞn
NMR spectroscopy
All samples were prepared in potassium phosphate buffer
10 mM, pH 6.0), 5% D O and 10% DMSO-d . NMR spectra of
C/ N labelled Fyn SH3 were collected at 25 °C on a 600 MHz
(
2
6
Acknowledgements
1
3
15
Varian INOVA spectrometer equipped with a cold probe This work was supported by the European Regional Develop-
1
5
(
Varian). Resonance assignments of Fyn SH3 for backbone
N
ment Fund (ERDF), ISCE-Chem INTERREG IV A France-
1
15
and HN nuclei were obtained by 2D H- N-HSQC, HNCA, (channel)-England project no. 4061. We also acknowledge
HNCACB and CBCA(CO)NH spectra incorporating water flip- Diamond Light Source for time on Beamline/Lab I19 under
back and gradient enhancement on a 1 mM protein sample. Proposal MT8521 and the Biophysics centre, University of
Assignments for peptide-bound Fyn SH3 were deduced by track- Southampton for CD spectroscopy. The authors also thank
ing peak movements in titration data. All peptide interaction Prof. Richard J. Whitby for DFT calculation, and Dr Patrick
2
4
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Duriez from the Cancer Research UK Protein Core Facility,
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2
2
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Org. Biomol. Chem., 2015, 13, 4562–4569 | 4569