Automated lineshape analysis of complex NMR spectra for a novel synthetic tetrafluorobisphosphonate, a potential ligand for phosphoglycerate kinase

Article abstract of DOI:10.1080/10426507.2015.1073281

Tetraalkyl 1,1,3,3-tetrafluoro-2,2-dihydroxypropane-1,3-bisphosphonates were prepared. The complex ³¹P{¹H}- and ¹⁹F-NMR spectra were analyzed as [[A]₂X]₂ and related systems. Modern methods of automated spectral analysis using DAISY under WIN-NMR were applied.

Full text of DOI:10.1080/10426507.2015.1073281

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Automated lineshape analysis of complex
NMR spectra for a novel synthetic
tetrafluorobisphosphonate, a potential ligand for
phosphoglycerate kinase
G. M. Blackburn, G. Hägele, A. Hottgenroth, A. J. Ivory, D. L. Jakeman & R.
Spiske
To cite this article: G. M. Blackburn, G. Hägele, A. Hottgenroth, A. J. Ivory, D. L. Jakeman
& R. Spiske (2016) Automated lineshape analysis of complex NMR spectra for a novel
synthetic tetrafluorobisphosphonate, a potential ligand for phosphoglycerate kinase,
Phosphorus, Sulfur, and Silicon and the Related Elements, 191:3, 367-372, DOI:
10.1080/10426507.2015.1073281
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Date: 30 March 2016, At: 00:49

PHOSPHORUS, SULFUR, AND SILICON
, VOL. , NO. , –
http://dx.doi.org/./..
Automated lineshape analysis of complex NMR spectra for a novel synthetic
tetrafluorobisphosphonate, a potential ligand for phosphoglycerate kinase
G. M. Blackburna, G. Hägeleb, A. Hottgenrothb, A. J. Ivorya, D. L. Jakemana,c, and R. Spiskeb
aDepartment of Molecular Biology, Krebs Institute, The University of Sheffield, Sheffield, UK; bInstitut für Anorganische Chemie und Strukturchemie,
Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany; cDepartment of Chemistry, College of Pharmacy, Dalhousie University, Halifax, Nova
Scotia, Canada
ABSTRACT
ARTICLE HISTORY
Received  May 
Accepted  July 
Tetraalkyl 1,1,3,3-tetrafluoro-2,2-dihydroxypropane-1,3-bisphosphonates were prepared. The complex
31P{1H}- and 19F-NMR spectra were analyzed as [[A]2X]2 and related systems. Modern methods of automated
spectral analysis using DAISY under WIN-NMR were applied.
KEYWORDS
Tetraalkyl ,,,-tetrafluoro-
,-dihydroxypropane-,-
bisphosphonates; synthesis;
NMR
GRAPHICAL ABSTRACT
of these molecules we generated the tetrafluorobisphosphonates
(3), which exhibit complex nuclear magnetic resonance (NMR)
spectra. Results from spectral analysis are presented in this
paper.
Introduction
Phosphonates have been recognized as biologically important
tools, which have lead to a greater understanding of biological
processes.1 Often their application has been as transition state
analogues for acyl transfer processes, and as ground state ana-
logues for phosphoryl transfer in enzymatic reactions. Their use
is based upon the principal fact that the deletion of, or replace-
ment of, the scissile P-O-C linkage with a non-scissile P-C linker
maintains the phosphoryl moiety within the molecule. Different
recognition processes are achieved within the enzyme active site.
Halogenation of the α-carbon has been proposed as an addi-
tional means of altering the recognition in enzyme processes2
as a result of improving the isosteric and isoelectronic nature of
the halogenated molecule.
Chemical discussion
The synthesis of (3) is outlined in Scheme 1. Dialkyl difluo-
romethanephosphonate (1) was obtained according to a mod-
ified literature preparation.5 Lithium diisopropylamide (LDA)
was used to deprotonate (1) and the anion reacted with CO2 to
synthesize dialkyl phosphonodifluoroethanoic acid6 (2). Appre-
ciable quantities of dihydroxybisphosphonate (3) were formed
during the reaction, which was isolated and recrystallized from
methylene chloride (DCM)/petrol. The 31P{1H} and 19F-NMR
spectra for the tetraethyl ester (3a) and the tetraisopropyl ester
(3b) are complex. In a subsequent section of this paper we
We have synthesized a range of α,α-difluoroalkylphos
phonates, which have been tested for their binding affinity
toward phosphoglycerate kinase.3,4 During the synthesis of one
CONTACT G. M. Blackburn
Sheffield S TN, UK.
g.m.blackburn@sheffield.ac.uk
Department of Molecular Biology, Krebs Institute, The University of Sheffield, Western Bank, Firth Court,
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
©  Taylor & Francis Group, LLC

368
G. M. BLACKBURN ET AL.
Scheme . Synthesis of compounds (1)–(3).
present the results from NMR spectral analysis for tetraiso- an AMX-500 MHz spectrometer was used. Toluene was dis-
propylester (3b).
tilled from calcium hydride, and tetrahydrofuran (THF) was dis-
Sodium dialkyl phosphite reacts with chlorodifluoromethane tilled from potassium and benzophenone. Column chromatog-
to form dialkyl difluoromethanephosphonate1 (1). Deprotona- raphy was performed using Kieselgel 60, 230–400 mesh (Merck
tion of (1) with LDA yields the corresponding carbanion, which 9835). All other chemicals were purchased from Aldrich Chem-
reacts with CO2 to form primarily the lithium salt of dialkyl ical Company Ltd.
phosphonodifluoroethanoic acid.2 A secondary nucleophilic
attack of carbanion produces the dilithium salt of tetraalkyl
1,1,3,3-tetrafluoro-2,2-dihydroxypropane-1,3-bisphosphonate.
Diethyl difluoromethanephosphonate5 (1a)
Hydrolysis of the reaction mixture converted lithium salts
into the corresponding phosphonodifluoroethanoic acid (2)
and tetraalkyl 1,1,3,3-tetrafluoro-2,2-dihydroxypropane-1,3-
bisphosphonate (3).
Diethyl phosphite (44 g, 0.32 mol, freshly distilled) was added
drop-wise at RT under argon to sodium spheres (7.8 g, 0.34 mol)
in toluene (250 mL, freshly distilled). The solution was further
heated at reflux for 1.5 h, cooled, and transferred to a dry flask by
cannula. Chlorodifluoromethane was bubbled through the solu-
tion overnight. After addition of water (300 mL), and extrac-
tion with CH2Cl2 (DCM, 4 × 300 mL), the organic layers were
combined and dried (MgSO4). The solvents were removed in
vacuo to yield a colorless oil, which was purified by silica gel
chromatography eluting with DCM. This gave (1a) as a color-
less mobile oil (27.5 g, 45.6%); bp 80–90°C/5-mm Hg (lit.1: bp
85.6–86.5°C/12-mm Hg); RF (DCM) 0.62; H (250 MHz, CDCl3)
The formation of a tertiary alcohol (4) via a corresponding
lithium salt (Scheme 2) was not observed.
No evidence was obtained that the dihydroxy compound (3)
was dehydrated in solution to the parent ketone, the tetraalkyl
1,1,3,3-tetrafluoro-2-oxopropane-1,3-bisphosphonate
(5)
(Scheme 3) as might be expected from literature.7
Experimental
2
2
All NMR spectra of synthesis samples were recorded on a 5.86 (1H, dt, JPH 28 Hz, JFH 48 Hz, CF2H), 4.25 (4H, dq,
BRUKER AC-250 MHz spectrometer unless specified, where 3JHH 7.0 Hz, 2 × CH2), 1.31 (6H, t, 3JHH 7.0 Hz, 2 × CH3); δP
Scheme . Formation of a tertiary alcohol is not observed.
Scheme . Formation of a keto form is not evident.

PHOSPHORUS, SULFUR, AND SILICON
369
2
–75°C under nitrogen. After 1 h, CO2 (dried over CaCl2) was
bubbled through the solution for 2 h at –75°C. The reaction was
then left for stirring overnight and allowed to warm to RT. The
reaction was quenched by cautious addition of water (150 mL)
and EtOAc (200 mL), and the layers were separated. The aque-
ous layer was acidified with 1 M H2SO4 (to pH 1), and extracted
with EtOAc (5 × 30 mL). The combined EtOAc layers were
dried (Na2SO4), and the solvent was removed in vacuo to give
a solid, which was recrystallized from petrol to give white crys-
tals (2b) (6.5 g, 62%); mp 36–38°C; RF (4% MeOH/DCM) 0.17;
(101.20 MHz, CDCl3) 5.38 (1 P, t, JPF 91 Hz); δF (188 MHz,
CDCl3) –137.8 (dd, 2JFH 48 Hz, 2JPF 91 Hz).
Diethyl phosphonodifluoroethanoic acid6 (2a) and
tetraethyl 1,1,3,3-tetrafluoro-2,2-dihydroxypropane-1,3-
bisphosphonate (3a)4
Diethyl difluoromethanephosphonate (1a, 4.7 g, 25 mmol) was
added dropwise to a stirred solution of LDA (2M solution in
heptane/THF, 18.0 mL, 35 mmol) in THF (30 mL, dry) at –70°C
under nitrogen. After 20 min, CO2 (dried over CaCl2) was bub-
bled through the solution for 45 min at –75°C. The reaction was
then left for stirring overnight and allowed to attain RT. The
reaction was quenched by cautious addition of water (100 mL)
and ether (100 mL), and the layers were separated. The aque-
ous layer was acidified with 1M H2SO4 (to pH 1), and extracted
with EtOAc (5 × 30 mL). The combined EtOAc layers were dried
(Na2SO4), filtered, and the solvent was removed in vacuo to give
δ
(250 MHz, CDCl3) 10.00 (1H, bs, CO2H), 4.70–4.90 (2H,
H
m, CH), 1.33 (12H, d, 3JHH 7.5 Hz, 4 × CH3); δ (101.20 MHz,
P
CDCl3) 1.6 (t, 2JPF 96 Hz); δ (235.19 MHz, CDCl3) –114.3 (d);
F
m/z (EI) 231 (M+).
The initial EtOAc layer was dried (MgSO4), filtered, and the
solvent was removed in vacuo to yield an off-white solid and was
recrystallized from DCM/petrol to yield (3b) as a fluffy white
powder (1.2 g, 10% based on remaining (1b)), mp 61–62°C
(Found: C, 37.95; H, 6.28. C15H30F4O8P2 requires C, 37.82%;
H, 6.35%); δ (250 MHz, CDCl3) 4.80 (4H, m, CH), 1.37 (24H,
H
(2a) as a yellow oil (2.7 g, 46.5%); RF (5% MeOH/DCM) 0.17; δ
H
d, 3JHH 7.5 Hz, CH3); δ (202.40 MHz, CDCl3) 2.7–4.7 (m); dF
P
(250 MHz, CDCl3) 9.45 (1H, bs, CO2H), 4.1–4.3 (4H, m, 2 ×
(235.19 MHz, CDCl3) –118.5 to –119.5 (m); m/z (CI) 402 (M+).
This product was subjected to extensive NMR spectral analytical
studies presented below.
CH2), 1.3 (6H, dt, 3JHH 7.5 Hz, 2 × CH3); δ 3.5 (t, 2JPF 97 Hz);
P
δ –117.3 (d); m/z (EI) 231 (M+).
F
The ether layer was dried (MgSO4), filtered, and reduced in
vacuo to yield an off-white oil, which solidified on standing and
was recrystallized from DCM/petrol to yield(3a) as a white crys-
talline solid (0.17 g, 6.0% based on remaining (1a)), mp 55–58°C
(Found: C, 31.43; H, 5.13. C11H22F4O8P2 requires C, 31.44%; H,
5.28%); δ (250 MHz, CDCl3) 4.25–4.45 (8H, m, 4 × CH2), 1.4
(12H, dt,H3JHH 7.5 Hz, 6 × CH2); δP (202.40 MHz, CDCl3) 4.5–
7.5 (m); δ (235.19 MHz, CDCl3) –121.8 to –122.5 (m); m/z (CI)
402 (M+)F.
NMR spectroscopic investigations
Experimental
A sealed sample of (3b) as a 5% solution in CDCl3 was
made in the standard freeze-pump-thaw technique. 31P{1H}-
MR, 81 MHz, and 19F-NMR, 188 MHz, spectra were measured
at ambient temperature using the BRUKER AM200SY spec-
trometer at Düsseldorf.
Diisopropyl difluoromethanephosphonate5 (1b)
Diisopropyl phosphite (16.6 g, 0.10 mol, freshly distilled) was
added drop-wise at RT under nitrogen to sodium spheres (2.3 g,
0.10 mol) in toluene (150 mL, freshly distilled). The solution was
further heated at reflux for 3 h and then cooled. Chlorodifluo-
romethane was bubbled through the solution for 1 h. After addi-
tion of water (200 mL) and extraction with DCM (3 × 200 mL),
the organic layers were combined, dried (MgSO4), and filtered.
The solvents were removed in vacuo to yield a colorless mobile
oil (21.0 g, 95%); bp 85–95°C/5-mm Hg (lit.1 bp 89–90°C/12-
The P{H}-NMR spectrum – the X-part of an [[A]X]
spin system
The experimental, proton-decoupled 81-MHz 31P{1H}-NMR
spectrum of tetraisopropyl 1,1,3,3-tetrafluoro-2,2-dihydroxy-
propanebisphosphonate, 5% (3b) in CDCl3, is shown in
Figure 1a.
The proton-decoupled 31P{1H}-NMR spectrum is consistent
with an [[A]2X]2 spin system (A = 19F, X = 31P) resulting either
from a free rotating propane skeleton or a fixed conformer with
two-fold symmetry as shown in a simplified Scheme 4.
Explicit rules for the spectral analysis of [[A]2X]2 spin sys-
tems were given by Harris and coworkers.8,9 We follow this
standard notation using the coupling constants and combined
parameters NPF, LPF, NFF, and LFF as shown in Table 1. The
experimental 81-MHz 31P{1H}-NMR spectrum of (3b) is sym-
metric to the central resonance frequency vP. Three strong and
relatively narrow lines form a triplet centered at vP and separated
mm Hg); RF (DCM) 0.60; δ (250 MHz, CDCl3) 5.80 (1H, dt,
H
2
3
2JPH 28 Hz, JFH 48 Hz, CH), 4.70–4.90 (2H, m, JHH 7.0 Hz,
2 × CH), 1.30 (12H, t, 3JHH 7.0 Hz, 4 × CH3); δ (101.20 MHz,
CDCl3) 3.44 (1 P, t, 2JPF 92 Hz); δ (235.19 MHz,PCDCl3) –138.0
F
(dd).
Diisopropyl phosphonodifluoroethanoic acid6 (2b) and
tetraisopropyl 1,1,3,3-tetrafluoro-2,2-dihydroxypropane-
1,3-bisphosphonate (3b)4
2
4
by NPF = JPF + JPF. Starting from the standard values8–13 for
4
4
2
4
2JFF, JFF, JPP, JPF, and JPF automated analysis and iteration
Diisopropyl difluoromethanephosphonate (1b, 10 g, 46 mmol) for the [[A]2X]2 spin system using WINDAISY14 running under
was added dropwise to a stirred solution of LDA (2-M solution WIN-NMR15 yielded the NMR parameters given in Table 1 and
in heptane/THF, 25.0 mL, 50 mmol) in THF (50 mL, dry) at a simulation shown in Figure 1b above.

370
G. M. BLACKBURN ET AL.
Table . Resonance frequencies ν and coupling constants [Hz] from WINDAISY
P
iterations based upon the experimental -MHz P{H}-MR spectrum of (3b). Spec-
tral half-width of NPF lines: HWNPF = . Hz. Spectral half-width of all other lines:
HWLPF = . Hz. Resonance frequency ν from -MHz F-NMR spectrum.
F
Parameters
Final
Error
vA
vX
JAX
JAX´
JAA´
JAA´´
νF = ν2 = ν3 = ν4 = ν5
νP = ν1 = ν6
− .
− .
n.i.
.
2
2
2
2
2JPF = J12 = J13 = J46 = J56
.
.
.
n.i.
4JPF
=
4J15
2
=
4J16
=
gem
2
4J23
=
4J24
.
gem
gem
2JFF = J23 = J45
− .
.
4
cis
4
cis
4 cis
J
=
J
=
J
or
.
FF
24
35
trans
4
trans
4
4
trans
J
=
J
=
J
34
FF
25
JAA´´´
JXX´
J
=
J
=
J
or
− .
.
.
4
trans
4
trans
4
trans
FF
25
34
4
cis
4
cis
4
cis
J
=
J
=
J
FF
24
35
4JPP
=
4J16
.
n.i.: Not iterated.
Figure . (a) Upper: Experimental -MHz P{H}-NMR spectrum of tetraisopropyl
,,,-tetrafluoro-,-dihydroxypropane-,-bisphosphonate (3b) (%, solution in
CDCl). (b) Lower: Calculated spectrum using data from Table .
Figure . Experimental -MHz F-NMR spectrum of tetraisopropyl ,,,-
tetrafluoro-,-dihydroxy-propanebisphosphonate (3b) (%, solution in CDCl).
Results from F-NMR – some models for more complex spin
systems
The situation in the proton-coupled total spin system is much
more complex as shown in Figure 2 and Scheme 5:
Scheme . The [[A]X] spin system.
Scheme . The total [[A]K[RS]] spin system in (3b).
Parameters given in Table 1 are consistent with results using
explicit rules for the spectral analysis of [[A]2X]2 spin systems
given by Harris and co-workers.8, 9 Attempts to fit the 31P{1H}-
MR spectrum by the more simple [A2X]2 system were unsuc-
cessful, as expected from published theory.8,9
The experimental 188-MHz 19F-NMR spectrum of (3b) is
symmetric with respect to the central frequency vF. Two strong
and relatively narrow lines (HWNF = 0.9 Hz) form a doublet
2
4
centered at vF and separated by NPF = JPF + JPF = 94.167 Hz.
Model 1
The F{H}-NMR spectrum – the A-part of an [[A]X] spin
system
Some tentative simulations and iterations using the simpli-
We were not able to record the proton decoupled 19F{1H}- fied approximation for the A-part of the [[A]2X]2 spin system
NMR spectrum, consequently the complementary A-part of the were not successful for both using either the LAO-PC15 or the
[[A]2X]2 spin system, potentially serving as a proof for results WIN-DAISY14,15 programs. The NPF-lines in the 19F-NMR
given in Table 1 was not accessible.
spectrum exhibit a more narrow spectral half-width (0.9 Hz)

PHOSPHORUS, SULFUR, AND SILICON
371
Model 2
The total [[A]2K[RS6]2]2, a 34-spin system, holds the addi-
tional contributions: K from the hydroxyl protons, while the iso-
propoxy groups give rise to the RS6 units.
Model 3
If JFH couplings involving the hydroxyl protons and the 4JPOCCH
couplings are zero, then a simplified spin system results are
shown in Scheme 6.
Scheme . The [[A]XR] spin system.
Corresponding WIN-DAISY simulations based upon data
derived from the proton-decoupled 31P{1H}-NMR spectrum
(see Table 1) were applied to the [[A]2XR2]2 model fixing JPOCH
to a tentative value of 5 Hz. Comparison with the experimen-
tal 188-MHz 19F-NMR spectra of (3b) in Figure 2 showed that
a typical broad line shape in the fluorine part is not explained
by POCH coupling effects. But clearly this additional coupling
affects the phosphorus part, as expected.
Figure . Flow chart diagram of PC-simulator program BLACKFILM.
Model 4
and high intensities, while the remaining lines are broader
(2.3 Hz) and less intense.
A useful auxiliary program BLACKFILM was designed,
which allows simulation of film-like sequences of [[A]2X]2
spectra using individual line widths for NPF- and the remain-
ing (LPF) lines. The basic principles of BLACKFILM are shown
in Figure 3.
A more likely model suggests the existence of proton fluorine
couplings JFH. This effect may be due to through bond inter-
action or via the formation of F···H-O bridges leading to FH-
coupling in an [[A]2KX]2 spin system as shown in Scheme 7.
Corresponding WIN-DAISY simulations varying JFH from
0–4 Hz were compared with the experimental 188-MHz
19F-NMR spectra of (3b) in Figure 2. Indeed, the typical broad
line shapes in fluorine signals are consistent with the presence
of F···H coupling effects as shown in Figure 4, justifying the
approximation by [[A]2KX]2. This additional coupling does not
affect the simulated phosphorus-NMR spectrum.
Directly accessible parameters: Resonance frequencies, NPF,
and spectral half-width of NPF-lines HWNPF were kept at fixed
values. The remaining parameters: LPF, NFF, and LFF were var-
ied from minimal to maximal values. The individual coupling
constants were generated according to:
1. JPF = (NPF + LPF)/2 JPF´ = (NPF – LPF)/2,
2. JFF = (NFF + LFF)/2 JFF´ = (NFF – LFF)/2,
Model 5
and fed to an efficient LAOCOON-type simulator MINILA.16, 17
In addition, the spectral half-width HWLPF of the remaining Another model suggests the existence of anisochronic fluorine
(non-NPF) lines was looped from a minimum to a maximum spins. This effect may be due to the formation of F···H-O bridges
value. Specific data for spectral half-width HWNF and HWLF leading to more fixed conformations responsible for the [ABX]2
were assigned and used in a line-specific Lorentz generator. The spin system shown in Scheme 7 above. Corresponding WIN-
resulting Lorentz spectra were displayed on screen, producing a DAISY simulations varying ꢀ = vA – vB from 0–30 Hz were
film-like sequence. Visual recognition (by comparison with the compared with the experimental 188-MHz 19F-NMR spectra
experimental spectrum) should lead in fortuitous cases to the of (3b) in Figure 2. This anisochrony does not affect the phos-
best NMR meter combination. But in vain: additional line split- phorus part, thus justifying the approximation by [ABX]2. But
tings occur in the experimental spectrum, not consistent with the typical broad line shape in the fluorine part and its split-
the simplified [[A]2X]2 model. Consequently, a more complex ting pattern are not sufficiently explained with the assumption
analysis of the system had to be adopted, as shown in Scheme 5. of anisochrony effects.

372
G. M. BLACKBURN ET AL.
Scheme . The [[A]KX] – induced by F-H bridges.
Figure . The closest approximation of the F-NMR spectrum of (3b) from a series of [[A ]KX] simulations with WIN-DAISY. For data, see Table . JFH =  Hz. Spectral
half-width =  Hz.
Conclusions
4. Jakeman, D. L., Ivory, A. J., Williamson, M. P., Blackburn, G. M. J. Med.
The application of WIN-DAISY has solved the measured
31P spectrum of tetraisopropyl 1,1,3,3-tetrafluoro-2,2-
dihydroxypropane-1,3-bisphosphonate, and has significantly
clarified the corresponding 19F spectrum. The results of the
model system are enhanced by introducing F···H-O bridges.
Such bridges have often been suggested as components of the
binding of fluorinated inhibitors to enzymes, although they
have been difficult to verify by direct structural methods.18 We
now provide a computational basis to support such claims. The
coupling constants determined in this work should facilitate
the analysis of spectra recorded from other synthetic polyflu-
orophosphonates. WIN-DAISY has proved to be a versatile
tool for simulations and iterations of complex NMR spectra.
It has coped with symmetric spin systems and has enabled
the development of an automated assignment of line-specific
half-widths for such systems.
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Oligo-Phosphonsäure- und Phosphinsäure-Derivaten an einem Propan-
Gerüst,” Dissertation, Universität Düsseldorf, 1992.
13. Hägele, G.; Engelhardt, M.; Boenigk, W. Simulation und Automatisierte
Analyse von Kernresonanzspektren; VCH-Verlag: Weinheim, Germany,
1987.
14. Weber, U.; Spiske, R.; Höffken, H.-W.; Hägele, G.; Thiele, H. Man-
ual und Program System, BRUKER Manual 1993, commercial prod-
uct, BRUKER Analytische Messtechnik Forchheim-Rheinstetten, Ger-
many.
Funding
15. Programs WIN-NMR and WINDAISY; commercial products,
BRUKER Analytische Messtechnik Forchheim-Rheinstetten, Ger-
many. For details see www.bruker.com.
16. Hägele, G.; Goudetsidis, S.; Höffken, H. W.; Lenzen, Th.; Spiske,
R.; Weber, U. Phosphorus, Sulfur, Silicon Relat. Elem., 1993, 77,
262.
G. Hägele acknowledges material support from the Fonds der Chemis-
chen Industrie and a grant from Academic Research Collaboration (British
Council/DAAD). G. M. Blackburn acknowledges financial support from
the BBSRC (to A. J. Ivory) and a Research Studentship (to D. L. Jakeman).
17. Hägele, G.; Spiske, R. BLACKFILM program; designed 1996, Univer-
sität Düsseldorf; unpublished work, full details available from Prof. G.
Hägele on request.
18. Jin, Y.; Bhattasali, D.; Pellegrini, E.; Forget, S. M.; Baxter, N. J.; Cliff, M.
J.; Blackburn, G. M.; Waltho, J. P. Proc. Natl. Acad. Sci. USA 2014, 111,
12384–12389.
References
1. Engel, R. Chem. Rev. 1977, 77, 349–367.
2. Blackburn, G. M. Chem. Ind. (London) 1981, 5, 134–138.
3. Blackburn, G. M.; Jakeman, D. L.; Ivory, A. J.; Williamson, M. P. Bioorg.
Med. Chem. Lett., 1994, 21, 2573–2578.