Anti-staphylococcal activity and β-lactam resistance attenuating capacity of structural analogues of (-)-epicatechin gallate

Article abstract of DOI:10.1016/j.bmcl.2011.09.116

We examined the impact of gradual removal of hydroxyl groups from the A- and B-rings of (-)-epicatechin gallate on antibacterial activity and oxacillin resistance attenuation of an epidemic strain of methicillin resistant Staphylococcus aureus. Removal of

Full text of DOI:10.1016/j.bmcl.2011.09.116

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6996–7000
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-staphylococcal activity and b-lactam resistance attenuating capacity
of structural analogues of (ꢀ)-epicatechin gallate
James C. Anderson a, , Robert A. McCarthy a, Sarah Paulin b, Peter W. Taylor b,
⇑
⇑
a Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK
b School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1 1AX, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We examined the impact of gradual removal of hydroxyl groups from the A- and B-rings of (ꢀ)-
epicatechin gallate on antibacterial activity and oxacillin resistance attenuation of an epidemic strain
of methicillin resistant Staphylococcus aureus. Removal of both hydroxyls from the B-ring effected a large
reduction in oxacillin MIC (from 512 to 0.25 mg/mL at a concentration of 12.5 mg/L); further hydroxyl
deletion of the A-ring reduced the oxacillin effect but increased intrinsic anti-staphylococcal activity
Ó 2011 Elsevier Ltd. All rights reserved.
Received 31 August 2011
Revised 27 September 2011
Accepted 27 September 2011
Available online 8 October 2011
Keywords:
MRSA
(ꢀ)-Epicatechin gallate analogues
b-Lactam antibiotics
Antibacterial chemotherapy
Antibiotic resistance
Extracts of the tea plant, Camellia sinensis, contain abundant
quantities of both galloyl and nongalloyl catechins; these polyphe-
nols constitute around 20% of the dry leaf weight and contribute to
the weak antibacterial activity of green tea preparations.1
Subinhibitory concentrations of these secondary metabolites exert
a range of effects on bacterial cells that have the potential to be
harnessed for therapeutic and industrial use. For example, moder-
ate concentrations of (ꢀ)-epicatechin gallate (ECg), and to a lesser
extent (ꢀ)-epigallocatechin gallate (EGCg) and (ꢀ)-catechin gallate
(Cg), disrupt the b-lactam-resistance machinery of methicillin
resistant strains of Staphylococcus aureus (MRSA), inducing com-
plete but reversible susceptibility to this important group of anti-
binding proteins responsible for peptidoglycan biosynthesis and
b-lactam resistance.4,7
The interaction of galloyl catechins with model phospholipid
membranes is partly governed by their relative hydrophobicity,
with ECg showing a higher affinity than EGCg. ECg penetrates to
a location deep within the phospholipid palisade8 and in bilayers
containing phosphatidylcholine (PC) there are strong initial cat-
ion-p interactions between the galloyl ring and quaternary amine
of the head group.9 ECg differs from EGCg only in the absence of a
hydroxyl function at one of the meta- positions of the B-ring (Fig. 1)
and we proposed,10 on the basis of enhanced microbiological activ-
ity of ECg, that a further reduction in the degree of B-ring hydrox-
ylation would enhance anti-MRSA effects by increasing the affinity
of these analogues for lipid bilayers. In addition B-ring 4-hydroxyl
deletion should also give compounds less prone to epimerization
via a quinone methide-like intermediate due to the removal of this
anchimeric assistance.10 We demonstrated that a monohydroxy-
lated 3-hydroxy B-ring 1 and a dihydroxylated 3,5-dihydroxy
B-ring 2 ECg analogue sensitised MRSA strains to the b-lactam
antibiotic oxacillin to a comparable extent compared to the natural
product (Fig. 1).10 In combination with hydrolytically more stable
analogues possessing an amide linked gallate group 3,11 these
modifications may provide a route to compounds of therapeutic
interest. We now report evaluation of the anti-MRSA activity of
further analogues of naturally occurring galloyl catechins with var-
iable degrees of hydroxylation of the A- and B-rings (Fig. 1). We
have synthesized, in enantiomerically pure form, the B-ring phenyl
analogue 4 and the corresponding A-ring free hydroxyl compounds
biotics.2
Galloyl catechins also
inhibit formation of
staphylococcal biofilms,3,4 reduce the secretion of toxins and other
virulence-related proteins by S. aureus strains5 and abolish
halotolerance in staphylococcal strains associated with food spoil-
age and food poisoning.6 Although the processes involved in these
phenotypic modifications are as yet incompletely defined, there is
strong evidence that they are dependent on the intercalation of the
bioactive polyphenols into the bacterial cytoplasmic membrane
(CM): the most potent modifier, ECg (Fig. 1), inserts into the staph-
ylococcal bilayer, inducing a series of complex changes to the
phospholipid palisade and leading to reduction in the efficiency
of function of CM-embedded proteins such as the penicillin
⇑
Corresponding authors.
E-mail address: j.c.anderson@ucl.ac.uk (J.C. Anderson).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.116

J. C. Anderson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6996–7000
6997
R
OH
OH
5
R'
4
3
B
B
O
HO
O
O
B
HO
O
A
A
R''
O
NH
O
O
OH
OH
OH
OH
OH
OH
OH
O
O
O
OH
OH
OH
O
6
3
5
R=H, R', R''=OH (-)-ECg
R, R', R''=OH, (-)-(EGCg)
1 R, R'=H, R''=OH
2 R, R''=OH, R'=H
4 R, R', R''=H
OH
OH
OH
OH
Figure 1.
in the epicatechin 5 and catechin 6 relative stereochemistry to
compare their capacity to suppress the resistance of MRSA isolates
to the b-lactam antibiotic oxacillin.
R
O
BnO
OH
i
iii
7
The enantiomer of 4 has previously been synthesized in unde-
termined enantiomeric purity by Chan and co-workers.12 In the
synthesis reported here the introduction of absolute and relative
stereochemistry is almost identical to the route developed by Chan
and co-workers,12 but we have quantified the enantioselectivity by
chiral HPLC with comparison to racemic standards.13 We also
chose to begin our synthesis in an analogous fashion to our synthe-
ses of 2 and 3,10 and this synthesis differs from Chan’s in the route
to the benzyl styrene 7 (Scheme 1). Aldol condensation of 910 with
acetophenone gave enone 10 in 70% yield (Scheme 1).14 As we had
noted before, reduction of this type of enone system to the satu-
rated alcohol and subsequent elimination to give the alkene was
surprisingly capricious.10 Reduction of the conjugated alkene with
catechol borane14 and subsequent reduction of the ketone with
lithium aluminium hydride gave the corresponding saturated alco-
hol in 61% yield over two steps. Dehydration by first bromination
and then elimination with DBU gave 7 in gram quantities in good
overall yield (Scheme 1).
OBn
ii
OH
11 R=MOM
12 R=OH
BnO
O
Ph BnO
O
Ph
BnO
OH
Ph
O
H
OH
O
H
OBn
iv,v
HO
OBn
14 3%
OBn
13 14%
15 6%
O
4
O
OH
OH
OH
O
OH
Control of absolute stereochemistry was achieved by enantiose-
lective Sharpless dihydroxylation that had previously been devel-
oped by Chan12 and which we had used successfully ourselves.10
Treatment of 7 using AD-mixbÒ15 gave 11 in 46% yield in an initial
76% ee as measured by chiral HPLC of the corresponding dimethyl
acetal (Scheme 2).16 Tritration of the solid with an Et2O/EtOAc mix
upgraded the material to 96% ee in 38% isolated yield. Removal of
the MOM ether gave triol 12 which contained ꢁ8% of a dimethyl
acetal impurity. This was removed by recrystallisation (Et2O) to
give pure 12 in 81% yield. This material corresponded to the inter-
mediate in Chan’s synthesis of the enantiomer of 4.12 Cyclisation to
the 2,3-cis-substituted C-ring followed the procedure used by Chan
that we had also used in our previous syntheses.10 In our hands
treatment of 12 with trimethyl orthoformate and then ring open-
ing with acetyl bromide17 gave a mixture of products. Optimisation
experiments led us to prepare acetyl bromide in situ from
Scheme 2. Reagents and conditions: (i) AD-mix-bÒ, t-BuOH, H2O, MeSO2NH2, 0 °C,
5 days, 46% @76% ee, 38% @96% ee; (ii) HCl, MeOH, Et2O, reflux, 6.5 h, 81%; (iii)
HC(OMe)3, PPTS cat., CH2Cl2, rt; w/up then AcBr, 4 Å mol sieves, CH2Cl2, rt; K2CO3,
acetone, rt, 48 h; w/up then NaBH4, MeOH, DME rt, 75 min, 13 14%, 14 3% and 15 6%
(over four steps); (iv) tri-OBn gallic acid chloride, DMAP, Et3N, CH2Cl2, rt, 16 h, 61%;
(v) H2, 10% Pd(OH)2/C, EtOAc, rt, 6 h, 89%.
phosphorus tribromide and acetic anhydride to minimise any
HBr formation. A small excess of acetic anhydride was used to
sequester any water present. Treatment of the mixture of alkyl
bromides with K2CO3/acetone to induce cyclisation and then re-
moval of the formate ester with sodium borohydride gave a diaste-
reomeric mixture of pyrans 13 (14%) and 14 (3%) and the
dihydrobenzofuran 15 (6%) (Scheme 2). The relative stereochemis-
try of 13 was assigned in part on the multiplicity of the 1H NMR
signal of the ArCHO proton in 14 (the corresponding signal in 13
is overlapping with PhCH2 signals) which has J = 7.9 Hz (d 4.79,
1H, d), indicative of a trans relationship of the two pyran substitu-
ents. The multiplicity of this signal in the final compound 4 also
correlates with other systems (vide infra). The relative stereochem-
istry of 15 is based on an assumed double inversion mechanism,
resulting in retention of stereochemistry in the formation of the
furan ring. The double inversion mechanism is widely accepted
to account for the retention of stereochemistry seen in the ex-
pected cyclisation to 13.17 However the isolation of a small amount
of diastereoisomer 14 suggests that epimerization of the interme-
diate bromide centre is possible at some point in the reaction se-
quence. Completion of the synthesis was achieved by DMAP
catalysed coupling with 3,4,5-tribenzoyloxy benzoic acid chloride
(61%) followed by global hydrogenolysis with Pd(OH)2 catalysis
BnO
OMOM
BnO
OMOM
O
ii-v
i
CHO
OBn
OBn
10
9
BnO
OMOM
7
OBn
Scheme 1. Reagents and conditions: (i) KOH aq (25%), PhCOMe, EtOH, THF, rt, 16 h,
70%; (ii) Catechol borane, THF, ꢀ78 °C to rt, 99% crude; (iii) LiAlH4, THF, 0 °C, 61%
crude; (iv) PPh3, Br2, Et3N, CH2Cl2, 0 °C to rt, 91%; (v) DBU, PhMe, 110 °C, 16 h, 81%.

6998
J. C. Anderson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6996–7000
decomposed when stored neat at ꢀ20 °C. Protection with MOMCl
OH
i
(99%) made the product 16 much more stable. The rest of the syn-
thesis followed the established route.10,12 Sharpless asymmetric
dihydroxylation with AD-mixbÒ15 gave 22 in 67% yield in 96% ee
as measured by chiral HPLC of the corresponding dimethyl acetal.16
Removal of the MOM group gave the triol 23 and subjection of this
to the optimised conditions for orthoformate formation, conver-
sion to the corresponding alkyl bromide, cyclisation and removal
of the formate esters gave a mixture of products. We isolated the
desired A-ring compounds 24/25 as an inseperable trans/cis mix-
ture in a ratio of 4:1, the desired product being the minor compo-
nent. Clearly the double inversion mechanism is breaking down in
these simpler unactivated benzylic cases. We also isolated a dihy-
drobenzofuran byproduct 26, as described above. Esterification of
the mixture 24/25 led to separable protected esters 27 (5%) and
28 (65%). Global hydrogenolysis gave the desired A-ring modified
compounds 5 (91%) and 6 (63%) in 96% ee.
O
O
ii
iii
20
19
OR
R
O
OH
vii
v
21 R=OH
16 R=MOM
iv
OH
22 R=MOM
23 R=OH
vi
O
Ph
O
Ph
OH
Ph
OH
OH
O
1 : 4
20%
H
25
24
26 4%
viii
The stereochemical assignment of the C2 and C3 substitution in
5 and 6 follows from the 1H NMR chemical shifts and multiplicities
of the ArCHO signal in each (5 d 5.43, 1H, s, and 6 d 5.41, 1H, d,
J = 6.1 Hz) which correlates with 4 and agrees with our previous
analysis.12 Absolute stereochemistry was assumed from the
Sharpless mnemonic for AD-mixbÒ.15
O
O
O
O
OR
OR
OR
OR
O
O
Minimum inhibitory concentration (MIC) of each compound
against the epidemic MRSA clinical isolate EMRSA-16 was deter-
mined by the CLSI (formerly NCCLS) broth microplate assay as pre-
viously described.2 For MIC determination, compounds were
dissolved in 60% v/v EtOH prior to dilution in broth; at the concen-
tration used the solvent had no effect on bacterial viability. The
capacity of the compounds to modulate the b-lactam resistance
of EMRSA-16 was evaluated by determination of the MIC at a fixed
concentration in combination with oxacillin. Assays were per-
formed in 96-well microtitre trays with a bacterial inoculum of
27 R=Bn
R=OH
28 R=Bn
ix
ix
5
6
R=OH
OR
OR
Scheme 3. Reagents and conditions: (i) PhOH, DIAD, Ph3P, PhMe. ꢀ25 °C, 73%; (ii)
H2, Lindlar cat., EtOH, rt, 86%; (iii) PhMe, 110 °C, 56%; (iv) NaHꢂMOMCl, THF, 99%;
(v) AD-mixb-Ò, t-BuOH, H2O, MeSO2NH2, 0 °C, 5 days, 67% @ 96% ee; (vi) HCl, MeOH,
Et2O, reflux, 7.5 h, 99%; (vii) HC(OMe)3, PPTS cat., CH2Cl2, rt; w/up then AcBr,
4 Å mol sieves, CH2Cl2, rt; K2CO3, acetone, rt, 48 h; w/up then NaBH4, MeOH,
t-BuOMe rt, 75 min, 24/25 20% and 26 4% (over four steps); (viii) tri-OBn gallic acid
chloride, DMAP, Et3N, CH2Cl2, rt, 16 h, 27 5%, 28 65%; (ix) H2, 10% Pd(OH)2/C, EtOAc,
rt, 5 4 h, 91%, 6 5 h, 63%.
around 104 colony-forming units in 100
ll of Mueller–Hinton
(MH) broth (Oxoid, Basingstoke, UK) supplemented with 2% w/v
(89%) to give 4 (96% ee). The stereochemical integrity of the 2,3-cis
substitution was proven from the multiplicity of the 1H NMR sig-
nal of ArCHO (d 5.31, 1H, br s) which agrees with our previous anal-
ysis12 of the relative stereochemistries of 1 and 2. Absolute
stereochemistry was assumed from the Sharpless mnemonic for
AD-mixbÒ15 and the fact that the optical rotation exhibits the same
sense of optical rotation as naturally occurring (ꢀ)-ECg.
NaCl. Doubling dilutions of oxacillin were used.
S. aureus EMRSA-16 is highly resistant to the b-lactam agent
oxacillin, with a MIC of 512 mg/L (Table 1). ECg possesses barely
discernable anti-staphylococcal activity against EMRSA-16 (Table
1) and other Gram-positive bacteria.1 However, the presence of
12.5 mg/L ECg in the oxacillin assay elicits a reduction in MIC to
1 mg/L, essentially abrogating the b-lactam resistance machinery
of EMRSA-16. This effect is not confined to EMRSA-16; in a previ-
ous study we showed that ECg is able to abolish resistance to oxa-
cillin and a range of other b-lactam drugs in forty MRSA strains
isolated from all major regions of the globe.2
The asymmetric synthesis of the novel A ring analogues 5 and 6
relied again on the Sharpless dihydroxylation/ortho-ester
cyclisation methodology that had previously been developed by
Chan12 and which we had used successfully ourselves.10 We inves-
tigated whether we could access the key alkene 16 using the Mits-
unobu chemistry also reported by Chan.12 Unfortunately in this
particular case treatment of phenol and cinnamyl alcohol with
di-isopropyl azodicarboxylate (DIAD) and triphenyl phosphine
led mainly to the direct substitution product 17 and the electro-
philic aromatic substitution product 18 (Eq. 1).
Compound 2 displayed an identical anti-EMRSA-16 profile to
ECg, indicating that reduction of B-ring substitution to a single
hydroxyl function at the 3-position did not influence
Table 1
Minimum inhibitory concentrations (MICs) and b-lactam modifying capacity of (ꢀ)-
HO
O
epicatchin gallate (ECg) and structural analoguesa
HO
OH
Ph
ð1Þ
Compound
MIC (l
g/mL)
Oxacillin MIC (
Compound (
lg/mL)
lg/mL)
DIAD, Ph3P, PhMe
17
Ph
17 : 1
18
-25 °C
12.5
6.25
Oxacillin
ECg
1
2
4
512
128
128
128
64
—
1
1
1
0.25
NGb
NGb
—
—
—
—
2
It turned out that the use of a propargyl alcohol circumvented
the selectivity issue producing the desired ether 19 as the main
product in 73% yield (Scheme 3). Classic Lindlar reduction of the al-
kyne 19 to the alkene 20 occurred uneventfully (86%) and required
refluxing in toluene for 48 h to promote a Claisen rearrangement to
deliver the required alcohol 21 in 56% yield. We noted that this
material was unstable for longer periods of heating and slowly
5
6
16
16
16
4
a
Assays were performed in duplicate and repeated; no variation was noted.
No growth.
b

J. C. Anderson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6996–7000
6999
anti-staphylococcal activity or its capacity to abrogate b-lactam
resistance (Table 1). Similarly, compound 3, with an unnatural 3,5-
dihydroxylation of the B-ring, is indistinguishable from ECg and 2
in these assays, strongly suggesting that these two analogues insert
into the staphylococcal cytoplasmic membrane phospholipid bi-
layer in a way comparable to ECg and we are currently investigating
biophysical aspects of membrane intercalation of these analogues.
However, the B-ring phenyl analogue 4 produced a more pro-
nounced, albeit still weak, anti-staphylococcal effect and displayed
an enhanced capacity to reduce oxacillin resistance in EMRSA-16
at a concentration of 12.5 mg/L. This latter result was unexpected,
as we had assumed that structural analogues of ECg would be unable
to reduce the oxacillin MIC below 1 mg/L, which we considered to
represent the limit of resistance abrogation with this compound
class.10,11 This is clearly not the case and analogue 4 provides an
opportunity to develop more potent resistance modifiers with no
significant bactericidal or bacteriostatic properties, a desirable fea-
ture when considering control of evolution and emergence of resis-
tance to therapeutic agents of this type.18
This initial data for analogue 4 suggests an increased affinity for
the staphylococcal membrane compared to ECg and compounds 1
and 2. This contention is supported by the capacity of 4 to
considerably enhance, over a range of concentrations, the oxacillin
resistance modifying capacity of ECg; in fact, appropriate
concentrations of ECg in combination with 4 reduce the MIC of EMR-
SA-16 to 0.125 mg/L (Fig. 2). Further reduction in hydroxylation by
deletion of the two hydroxyl moieties on the A-ring of the B-ring
phenyl analogue, yielding 5, appeared to alter the nature of the inter-
action with the staphylococcal membrane; a substantial increase in
anti-staphylococcal activity was evident, accompanied by a reduc-
tion in b-lactam resistance modifying potential (Table 1). The Cg
analogue 6 showed a similar profile but exhibited an enhanced resis-
tance modifying effect at a concentration of 6.25 mg/L compared to
the ECg analogue 5. This contrasts with the resistance modifying
capacity of the corresponding natural products; ECg is a significantly
more potent modifier than Cg.2 Due to the increased bacterial
growth inhibiting activity of 5 and 6 (Table 1), these compounds
could not be examined at concentrations above 6.25 mg/L. Thus, in-
creases in the lipophilicity of domains governed by the
A- and B-rings result in the appearance of a more overt anti-staphy-
lococcal effect, in all probability due to damage to the cytoplasmic
membrane, rather than the more subtle effects that result from
membrane intercalation of the natural products.4,7
Based upon our observation that reduction of B-ring hydroxyl-
ation in the series of compounds EGCg, ECg, 1 and 2 enhanced
anti-MRSA effects, we synthesized, in single enantiomer form, the
epicatechin gallate analogue with free B-ring hydroxylation 4 and
the corresponding A-ring free hydroxyl compounds in the epicate-
chin 5 and catechin 6 relative stereochemistry (Fig. 1). The introduc-
tion of absolute chirality relied upon the asymmetric Sharpless
dihydroxylation reaction and the creation of the C-ring relative ste-
reochemistry by an ortho-ester/bromide cyclisation strategy. Previ-
ously this strategy has been shown to give good compound yields.
However, the lack of electron-releasing hydroxyl groups in the A
and B rings compromised the efficiency of the cyclisation strategy
in the cases presented here. There is a clear need for alternative syn-
thetic routes to deliver these compounds and other analogues which
will be the subject of our future work. Biological assays showed that
4 displayed an enhanced capacity to reduce oxacillin resistance in
EMRSA-16 at a concentration of 12.5 mg/L, reducing the oxacillin
MIC from 512 to 0.25 mg/L. Complete deletion of A and B ring hydro-
xyl groups in either epicatechin or catechin stereochemistry re-
sulted in a reduction in b-lactam resistance-modifying potential
and an increase in intrinsic anti-staphylococcal activity (Table 1).
These results support our hypothesis that reduction of B-ring
hydroxylation enhances intercalation of the epicatechin B-ring into
the staphylococcal cytoplasmic membrane, compromising the b-
lactam resistance machinery. Ongoing work is aimed at the design
and characterisation of ompounds of therapeutic potential inspired
by these results and by membrane intercalation studies.
Figure 2. Effect of combinations of ECg and 4 on the minimum inhibitory concentration (MIC) of oxacillin for EMRSA-16.

7000
J. C. Anderson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6996–7000
7. Bernal, P.; Lemaire, S.; Pinho, M. G.; Mobashery, S.; Hinds, J.; Taylor, P. W. J. Biol.
Acknowledgments
Chem. 2010, 285, 24055.
8. Caturla, N.; Vera-Samper, E.; Villalaín, J.; Reyes Mateo, C.; Micol, V. Free Rad.
Biol. Med. 2003, 34, 648.
9. Uekusa, Y.; Kamihira-Ishijima, M.; Sugimoto, O.; Ishii, T.; Kumazawa, S.;
Nakamura, K.; Tanji, K.; Naito, A.; Nakayama, T. Biochim. Biophys. Acta 2011,
1808, 1654.
This work was supported by Research Grant G0801757 from the
Medical Research Council. We thank Mr. J. Hill and Dr. L. Harris for
providing mass spectra and Ms. G. Maxwell for microanalytical
data.
10. Anderson, J. C.; Headley, C.; Stapleton, P. D.; Taylor, P. W. Tetrahedron 2005, 61,
7703.
11. Anderson, J. C.; Headley, C.; Stapleton, P. D.; Taylor, P. W. Bioorg. Med. Chem.
Lett. 2005, 15, 2633.
Supplementary data
12. Wan, S. B.; Landis-Piwowar, K. R.; Kuhn, D. J.; Chen, D.; Dou, Q. P.; Chan, T. H.
Bioorg. Med. Chem. 2005, 13, 2177.
13. See Supplementary data.
14. Van Rensburg, H.; Heerden, P. S.; Ferreria, D. J. Chem. Soc., Perkin Trans. 1 1997,
3415.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.116.
15. Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev. 1994, 94, 2483.
16. Enantiomeric purity measured by HPLC analysis of the corresponding dimethyl
acetal (see Supplementary data) against a racemic sample on a Chiralcel OD
column hexane/i-PrOH (90:10): 12 0.5 mL minꢀ1, R,R (major) 12.8 min, S,S
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14.3 min. 23 0.1 mL minꢀ1
, R,R (major) 4.4 min, S,S 5.7 min Absolute
configuration was assumed from the Sharpless mnemonic (see Ref. 16).
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pp 43–78.
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