Synthesis and phytotoxicity of structural analogues of thaxtomin natural products

Article abstract of DOI:10.1071/CH10006

Structural analogues of the phytotoxic thaxtomin natural products have been synthesized by building upon a piperazinedione core and from l-phenylalanine. The compounds were evaluated for their phytotoxic activity against Arabidopsis thaliana seedlings and some of the key features for activity have been identified. CSIRO 2010.

Full text of DOI:10.1071/CH10006

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2010, 63, 813–820
Synthesis and Phytotoxicity of Structural Analogues
of Thaxtomin Natural Products
Peter P. Molesworth,A Michael G. Gardiner,A Roderick C. Jones,A
Jason A. Smith,A,C Robert S. Tegg,B and Calum WilsonB
ASchool of Chemistry, University of Tasmania, Private Bag 75, Hobart, Tas. 7001, Australia.
BTasmanian Institute of Agricultural Research, University of Tasmania,
New Town Research Laboratories, 13 St Johns Avenue,
New Town, Tas. 7008, Australia.
CCorresponding author. Email: Jason.Smith@utas.edu.au
Structural analogues of the phytotoxic thaxtomin natural products have been synthesized by building upon a piperazine-
dione core and from l-phenylalanine. The compounds were evaluated for their phytotoxic activity against Arabidopsis
thaliana seedlings and some of the key features for activity have been identified.
Manuscript received: 4 January 2010.
Manuscript accepted: 17 March 2010.
NO2
O
O
Introduction
H
N
Me
N
R
The thaxtomins are a class of cyclic dipeptide derived com-
pounds containing a piperazinedione (also commonly referred
to as a diketopiperazine) core. They are produced by plant
pathogenic Streptomyces spp. responsible for common scab
disease;oneofthemosteconomicallysignificantdiseasesworld-
wide for potatoes.[1] Two representative examples, thaxtomin
A (1) and thaxtomin D (2), which differ mainly in hydroxylation
on the piperazine ring, are shown in Fig. 1. For the pathogen,
thaxtomin production is essential for disease transmission.[2]
These compounds are believed to inhibit cellulose deposition[3,4]
duringcellwallformationandarethoughttoinduceprogrammed
cell death[5] in picomolar concentrations. As part of a study to
develop new thaxtomin-tolerant strains of potatoes, we sought
to synthesize the natural products, as well as various analogues
for testing, to develop a greater understanding about structure
activity relationships. A previous model study targeted the syn-
thesis of thaxtomin A, and resulted in the preparation of the
de-nitro derivative via alkylation of a N,Nꢀ-dimethylbenzylidine
piperazinedione.[6] Herein we report the synthesis and phyto-
toxic activity of dehydro-structural analogues of the thaxtomins.
N
H
Me
R
R ꢀ OH, thaxtomin A (1)
R ꢀ H, thaxtomin D (2)
Fig. 1. Representative thaxtomin phytotoxins.
Aqueous workup gave the mono acetyl derivative 5. Subequent
hydrogenation[9] in a non-nucleophilic solvent (either THF or
ethyl acetate) with acetic acid to avoid catalyst poisoning, gave
7a and 7b, with the latter arising from migration of the acetyl
group in solution. The compounds could be readily separated by
1
chromatography and differ in the H NMR for the methylene
adjacent the phenyl group. In 7a these protons resonate as two
clearly defined doublet of doublets; while in the previously unre-
ported 7b, these protons appear as a multiplet. The assignment
of 7a is in excellent agreement with that previously reported,[10]
although the melting point obtained was 15◦ higher than litera-
ture. Further evidence for 7b being a structural isomer of 7a was
obtained by conversion of the individual compounds to the N,Nꢀ-
diacetylpiperazinedione 8 by reaction with acetic anhydride.The
hydrogenation of 5 required a non-nucleophilic solvent as ring
opening to give acetamide 6 was observed when methanol was
used (Scheme 1).
From compound 8 a second aldol condensation, using
cesium carbonate/DMF with the Boc-protected indole-3-
carbaldehyde[11] as a model, gave 9 which contains the carbon
skeleton of the thaxtomin but lacks the nitro group on the
indole. The Z geometry was confirmed for the alkene by X-ray
crystal structure determination of compound 9 (Fig. 2). The
acetyl and Boc-protecting groups could be removed by reaction
with an excess of hydrazine hydrate[6] to give thaxtomin-like
derivative 10.
Results and Discussion
The synthesis of the analogues was approached by building
upon the piperazinedione core of glycine anhydride 3, as this
would allow greater flexibility to introduce different substituents
onto either side of the molecule via aldol condensation chem-
istry. Thus, glycine anhydride (3) was activated by reaction
with acetic anhydride[7] to give diacetyl derivate 4, which was
then condensed with benzaldehyde. We found the use of a
modified method[8] using potassium carbonate in DMF prefer-
ential to triethyl amine/DMF[9] or t-BuOK/t-BuOH[10] systems
to introduce the phenyl substituent; and while cesium carbon-
ate has been reported,[8] it gave no significant improvement.
© CSIRO 2010
10.1071/CH10006
0004-9425/10/050813

RESEARCH FRONT
814
P. P. Molesworth et al.
O
N
O
O
O
O
O
O
X
R
R
Ac
H
(vi)
N
N
N
(iv)
(ii)
N
R
N
Ph
N
Ph
Ph
N
Rꢁ
Ac
H
N
O
Boc
5
X ꢀ H, 9
X ꢀ NO2, 11
Rꢁ ꢀ Ac, R ꢀ H, 7a
Rꢁ ꢀ H, R ꢀ Ac, 7b
R ꢀ Rꢁ ꢀ Ac, 8 (vii), (viii), (ix)
R ꢀ H, 3
R ꢀ Ac, 4
(i)
(v)
(iii)
(x)
O
X
O
O
NO2
O
O
H
AcHN
MeO
H
N
Me
N
N
Ph
N
Ph
N
Ph
N
H
H
Me
N
H
6
O
12
X ꢀ H, 10
X ꢀ NO2, 13
Scheme 1. Reagents and conditions: (i) ref. [2]; (ii) (a) PhCHO, K2CO3, DMF; (b) H2O; (iii) H2, 10% Pd/C, MeOH; (iv) H2,
10% Pd/C, THF, acetic acid; (v) Ac2O, Py, DMAP, CH2Cl2; (vi) (a) N-Boc-4-X-indole-3-carbaldehyde, Cs2CO3, DMF; (b) H2O;
(vii) 1 equiv. NH2NH2·H2O, DMF; (viii) MeI, K2CO3, DMF; (ix) and (x) excess NH2NH2·H2O, DMF.
been previously prepared, however attempts to do this selectively
in the presence of the nitro group, by reaction with magnesium
in methanol, borohydrides, or triethylsilane, were unsuccessful.
After initial phytotoxicity testing, vide infra, we sought
to synthesize the enantiomers of 12 and 13 derived from
l-phenylalanine. Thus the dipeptide 15, from the methyl ester
of l-phenylalanine (14), and a protected glycine was synthe-
sized in good yield using EDCI as the coupling agent.[13] The
diketopiperazine was formed in 73% yield by in situ ther-
mal deprotection[14,15] and cyclization, by heating the protected
dipeptide 15 in a sealed tube in a microwave reactor at 160◦C.
The cyclic dipeptide 16 was then activated with acetic anhy-
dride in refluxing dichloromethane. The choice of the base,
triethylamine instead of pyridine, was critical to achieving high
yields. The N,Nꢀ-diacetylpiperazinedione (3S)-8 was subjected
to the same synthetic sequence described for compound 7 to
give the derivatives (3S)-12 and (3S)-13. It is worth noting that
structure (3S)-12 has been previously reported in the patent
literature, as a potential herbicide,[16] by an aldol from the N,Nꢀ-
dimethylpiperazinedione but as such, no details were reported.
The intermediate (5S)-11 was a crystalline solid and X-ray
crystallography confirmed the formation of the Z alkene. How-
Fig. 2. X-ray crystal structure of 9; Molecules form centrosymmet-
ever, the crystal that was selected for analysis was racemic
(Fig. 3), indicating some racemization had occurred. It has been
reported that epimerization occurs during aldol condensations of
these activated piperazinediones, up to 30% in some cases,[17]
although these typically use much stronger bases such as LDA
or potassium t-butoxide. However, analysis of the literature val-
ues of the optical rotation of the two preceding intermediates
suggests that epimerization may in fact occur during diacetyla-
tion. While (3S)-8 has been reported numerous times, the optical
rotation has only been reported once, with a value of +7.8◦.[18]
In our hands, we observe a value of +19.2◦. The previously
reported method[18] to synthesize (3S)-8 involves heating with
sodium acetate in acetic anhydride at 100◦C, while we have used
milder conditions. It is thus possible that epimerization occurs
in the activation and not during the aldol condensation. As the
optical rotation is performed in methanol, it is possible that
deacetylation could result in formation of 16, which has a higher
ric dimers through H-bonding involving the amide N-H and the adjacent
carbonyl oxygen centre.
With the model study completed we then reacted 8 with the
N-Boc-4-nitroindole-3-carbaldehyde, prepared in four steps
from 2-methyl-3-nitroaniline using a modification of the
Bergman 4-nitroindole synthesis,[12] followed by a Vilsmeier–
Haack formylation and N-Boc protection (see Accessory Publi-
cation).The condensation gave compound 11, which was reacted
with one equivalent of hydrazine hydrate to selectively remove
the acetyl group. Reaction with methyliodide and potassium
carbonate in DMF, methylated the two nitrogen atoms of the
piperazinedione ring, and subsequent removal of the remain-
ing protecting group with excess hydrazine hydrate gave the
desired compound 12. If compound 11 is reacted with excess
hydrazine hydrate both the acetyl and Boc-groups are removed
to give 13. Compounds 12 and 13 are structural analogues of
thaxtomin D and des-N-methyl-thaxtomin C, compounds that
differ only by the presence of an alkene between the indole and
central piperazinedione rings. It should be noted that reduction
of the alkene would give the two natural products, which have not
1
optical rotation. However, analysis of this sample by H NMR
indicated no deacetylation had occurred. The reported optical
rotation for 16 also varies, with representative literature val-
ues including +97.8◦,[19] +60◦,[18] +26.6◦,[20] and +7.3◦;[15]
while we observed a value of +48.4◦. The cyclization to form

RESEARCH FRONT
Synthesis and Phytotoxicity of Structural Analogues of Thaxtomin Natural Products
815
Phytotoxicity
With the three structural variants synthesized, the phytotoxi-
city was evaluated in a bioassay using Arabidopsis thaliania
(Columbia) seedlings as the test subject. Arabidopsis thaliana
seeds were germinated on agar plates incorporating one of four
different concentrations (0.1 µM, 1.0 µM, 10.0 µM, 50.0 µM)
of the subject compound. These were each compared with a
healthy control. Thaxtomin A (isolated from S. scabiei broth)
was used for comparison. In each experiment, root length was
measured after 7 days of growth. Compound 10, which lacks
the nitro group, was inactive. Encouragingly, the racemic nitro-
containing derivatives 12 and 13 inhibited root growth by >80%
compared with the control, even at sub-micromolar concen-
tration (Fig. 4). Following this initial screen, a more detailed
evaluation was carried out to determine LD50 of the compounds,
as well as those derived from l-phenylalanine.
Compounds 12, 13, (3S)-12, (3S)-13, and thaxtomin A were
evaluated between 0.01 and 5.0 µM and the LD50 determined
(Fig. 5). A clear enhancement in activity for the methylated
derivative was observed and, surprisingly, the racemic 12 had
an LD50 of 0.143 µM, compared with 0.039 µM for the nat-
ural product thaxtomin A 1, and 0.699 µM for compound 13.
The enantio-enriched (3S)-12 was 25% (0.110 µM) more active
than the racemic derivative, not surprisingly indicating that the
enantiomer with the S configuration is more active than the R
configured isomer. These results suggest the active site must
have some degree of flexibility, as the conformational change
induced by introducing an alkene into the molecule does not
substantially alter the toxicity, whereas the stereochemistry at
the 3-position is clearly important.
Fig. 3. X-ray crystal structure of racemic 11; molecules form centrosym-
metric dimers through H-bonding involving the amide N-H and the adjacent
carbonyl oxygen centre.
This study has provided histological evidence to suggest these
compounds may inhibit plant cell development in a similar man-
ner as thaxtomin A. Thaxtomin A typically induces cell swelling
(hypertrophy), and enhances root hair proliferation in treated
A. thaliana shoot and root tissues (Fig. 6, panel B). Compounds
12 and 13 induced similar morphological changes in A. thaliana
seedlings (Fig. 6, panels D and C).
O
H
N
NH2
H
(i)
Boc
NH
H
Ph
14
H3CO
H3CO
Ph
15
O
O
(ii)
14
Conclusions
NO2
O
O
O
O
R
We have synthesized two structurally similar analogues of thax-
tomin that each exhibit potent phytotoxicity, inhibiting plant
growth and development in a similar manner to thaxtomin A.
Evidently, there is some conformational freedom in the active
site.Alkylation of the nitrogens of the central ring was associated
with enhanced activity.
(iii), (iv)
N
H
NH
N
Ph
HN
Ph
N
H
R
H
16
R ꢀ CH3, (3S)-12
R ꢀ H, (3S)-13
Scheme2. (i)EDCI, NEt3, Boc-Gly-OH, CH2Cl2;(ii)H2O, EtOH, 160◦C,
µ-wave; (iii) Ac2O, NEt3, DMAP, CH2Cl2,; (iv) as per conditions v through
x from Scheme 1.
Experimental
General Experimental
See Accessory Publication.
(3Z)-1-Acetyl-3-benzylidene-piperazine-2,5-dione (5)
piperazinediones under neutral or acidic conditions does not usu-
ally cause racemization, therefore it is likely to occur duirng
diacetylation or aldol condensation. Analysis by chiral chromo-
tography at each synthetic stage, is required to trace the source
of the racemization in these synthetic sequences. Attempts to
resolve the two enantiomers of 12 using NMR chiral shift
reagents to determine the extent of epimerization was unsuc-
cessful. While the compound is enriched in one enantiomer, as
indicated by the optical rotation ([α]2D0 = −23.1◦ (DMSO)), the
extent was not ascertained, as no data was reported for (3S)-12
(Scheme 2).[16]
To a solution of benzaldehyde (2.80 mL, 27.5 mmol) and 1,4-
diacetylpiperazine-2,5-dione 4 (4.951 g, 25.0 mmol), in DMF
(50 mL) under an atmosphere of nitrogen, was added potassium
carbonate (4.325 g, 31.2 mmol).The reaction was stirred for 18 h
before the addition of water. The resulting precipitate was col-
lected by filtration, washed with water, and dried under high
vacuumtoyieldthetitlecompound asanoff-whitesolid(5.549 g,
91%), mp 198–200◦C (lit.[9] 199–200◦C). νmax (solid/cm−1):
681, 768, 999, 1034, 1099, 1203, 1224, 1264, 1359, 1405, 1428,
1494, 1631, 1678, 1698, 3048, 3267. δH (CDCl3): 2.66 (s, 3H),

RESEARCH FRONT
816
P. P. Molesworth et al.
120
0.1 µM
1.0 µM
10.0 µM
50.0 µM
100
80
60
40
20
0
ꢂ10
ꢂ13
ꢂ12
TxA
Compound number
Fig. 4. Phytotoxicity screening results for 10, 12, 13, and thaxtomin A (TxA). Results shown are root length as a
percentage of the untreated control. Error bars indicate standard error.
120
110
100
90
80
70
60
50
40
30
20
10
0
(a)
(b)
(c)
(d)
Fig. 6. Seedlings from extended toxicity trials, at 80× magnification.
(a) Untreated control; (b) thaxtomin A treatment at 0.05 µM; (c) 13 at
2.0 µM; (d) 12 at 0.2 µM. Photographs were obtained using a Leica Dis-
ectting microscope MZ16 fitted with a Nikon Digital Sight DS-Fi1 (Nikon
Corporation, Japan).
(20 mg) under an atmosphere of hydrogen (1 atm, balloon) for
24 h. The solution was filtered through a plug of celite and
the solvent removed under reduced pressure, to yield the title
compound as a colourless glassy solid (0.233 g, 98%), mp 128–
130◦C. νmax (thin film/cm−1): 669, 1030, 1214, 1279, 1495,
1564, 1729, 1752, 1952, 3063, 3244, 3264. δH (CDCl3): 1.98 (s,
3H), 3.04 (dd, J 13.9, 6.2 Hz, 1H), 3.14 (dd, J 13.9, 6.2 Hz, 1H),
3.70 (s, 3H), 3.76–3.96 (m, 2H), 4.77–4.86 (m, 1H), 6.60 (bs,
1H), 6.90 (d, J 7.8 Hz, 1H), 7.10 (add, J 7.7, 1.5 Hz, 2H), 7.17–
7.30 (m, 3H). δC (CDCl3): 22.9, 37.8, 43.1, 52.5, 53.4, 127.2,
128.7, 129.3, 135.8, 168.9, 170.8, 172.0. m/z (EI+): 278 (M+,
<1%), 162 (100), 120 (35), 100 (38), 91 (20). HRMS calc. for
C14H18N2O4 [M+•] 278.12666, found 278.12659.
0.01
0.1
1
10
Concentration [µM] on a log scale
(3S)-13 (3S)-12 TxA ꢂ13
ꢂ12
Fig. 5. Comparison of the ability of compounds 12, 13, (3S)-12, (3S)-13 to
thaxtomin A to inhibit root growth in Arabidopsis thaliana seedlings. Error
bars indicate standard error.
4.51 (s, 2H), 7.18 (s, 1H), 7.32–7.54 (m, 5H), 7.97 (bs, 1H). δC
(CDCl3): 27.4, 46.3, 120.2, 125.8, 128.7, 129.6, 129.8, 132.7,
160.2, 162.9, 172.7. m/z (EI+): 244 (M+, 100), 202 (98), 173
(38), 117 (60), 91 (22). HRMS calc. for C13H12N2O3 [M+•
244.08479, found 244.08447.
]
( )-1-Acetyl-3-benzyl-piperazine-2,5-dione (7a)
and ( )-1-Acetyl-6-benzyl-piperazine-2,5-dione (7b)
( )-N-Acetylglycylphenylalanine Methyl Ester (6)
(3Z)-3-Benzylidenepiperazine-2,5-dione (200 mg, 0.819 mmol)
(3Z)-3-Benzylidenepiperazine-2,5-dione (4.629 g, 18.9 mmol)
was dissolved in methanol (20 mL) and stirred with 10% Pd/C
was dissolved in THF (400 mL)/acetic acid (40 mL), 10% Pd/C

RESEARCH FRONT
Synthesis and Phytotoxicity of Structural Analogues of Thaxtomin Natural Products
817
(400 mg) was added and the mixture stirred at room temperature
overnight, under an atmosphere of hydrogen (1 atm, balloon).
The residue was filtered through celite with ethyl acetate and the
solvent removed under reduced pressure. The residue was puri-
fied by elution from a short silica plug with dichloromethane
to yield ( )-1-acetyl-6-benzyl-piperazine-2,5-dione as a colour-
less crystalline solid (2.215 g, 48%). Flushing the plug with ethyl
acetate yielded ( )-1-acetyl-3-benzyl-piperazine-2,5-dione as
colourless crystalline solid (1.672 g, 36%) (84% combined
yield).
( )-1-Acetyl-3-benzyl-piperazine-2,5-dione (7a). mp 151–
153◦C (lit.[9] 136–138◦C). νmax (thin film/cm−1): 1097, 1205,
1258, 1367, 1436, 1684, 1704, 3246. δH (CDCl3): 2.59 (s, 3H),
3.12 (dd, J 13.9, 7.2 Hz, 1H), 3.26 (dd, J 13.9, 4.1 Hz, 1H), 3.47
(d, J 18.2 Hz, 1H), 4.21 (d, J 18.2 Hz, 1H), 4.37 (ddd, J 7.2, 4.1,
2.6 Hz, 1H), 6.53 (bs, 1H), 7.15–7.21 (m, 2H), 7.30–7.36 (m,
3H). δC (CDCl3): 27.4, 40.3, 45.6, 58.1, 128.2, 129.4, 129.7,
134.5, 166.4, 168.4, 171.9. m/z (EI+): 264 (M+, 11%), 204 (9),
91 (100). HRMS calc. for C13H14N2O3 [M+•] 246.10044, found
246.10049.
130.0, 134.6, 166.3, 168.3, 171.3, 171.5. m/z (EI+): 288 (M+,
20%), 246 (33), 203 (11), 131 (35), 91 (100). HRMS calc. for
C15H16N2O4 [M+•] 288.11101, found 288.11110.
( )-t-Butyl-3-[(Z)-(4-acetyl-5-benzyl-3,6-dioxo-piperazin-
2-ylidene)methyl]indole-1-carboxylate (9)
A solution of ( )-1,4-diacetyl-3-benzyl-piperazine-2,5-dione
(7) (0.803 g, 2.78 mmol), N-Boc-indole-3-carboxaldehyde
(0.751 g, 3.06 mmol), andcesiumcarbonate(1.273 g, 3.91 mmol),
indryDMF(25 mL)underanatmosphereofnitrogen, wasstirred
for 18 h protected from light. The reaction was quenched with
water (30 mL), and extracted with ethyl acetate (3 × 75 mL).The
combined organic layers were washed with water (2 × 75 mL)
and brine (2 × 70 mL), dried on magnesium sulfate, filtered and
the solvent removed under reduced pressure, yielding the title
compound as a solid which was used without further purifica-
tion, mp 181–183◦C dec. (1.084 g, 94%). νmax (solid/cm−1):
700, 750, 1091, 1150, 1221, 1309, 1336, 1368, 1453, 1631,
1677, 1712, 1735, 2985, 3155, 3256. δH (d6-DMSO): 1.67
(s, 9H), 2.49 (s, 3H), 3.07 (at, J 5.2 Hz, 2H), 5.08 (at, J
5.0 Hz, 1H), 6.69 (s, 1H), 6.96–7.14 (m, 5H), 7.31 (at, J
7.3 Hz, 1H), 7.38 (at, J 7.6 Hz, 1H), 7.58 (d, J 7.9 Hz, 1H),
7.99 (s, 1H), 8.03 (d, J 8.4 Hz, 1H), 10.35 (bs, 1H). δC (d6-
DMSO): 27.5, 28.3, 38.9, 58.1, 85.3, 110.0, 113.2, 115.5,
119.7, 123.9, 125.7, 126.6, 127.3, 127.7, 128.9, 130.5, 134.6,
135.4, 139.1, 149.5, 161.8, 166.3, 172.4. m/z (EI+): 473 (M+,
<1%), 373 (30), 331 (13), 240 (100), 212 (15), 155 (45).
( )-1-Acetyl-6-benzyl-piperazine-2,5-dione (7b). νmax (thin
film/cm−1): 703, 1097, 1205, 1258, 1367, 1439, 1680, 1699,
3254. δH (CDCl3): 2.56 (s, 3H), 3.18 (m, 2H), 3.24 (d, J 18.3 Hz,
1H), 4.18 (d, J 18.3 Hz, 1H), 4.39 (td, J 5.4, 2.6 Hz, 1H), 7.16–
7.19 (m, 2H), 7.29–7.38 (m, 3H), 7.39 (bs, 1H). δC (CDCl3):
27.4, 40.4, 45.4, 58.2, 128.1, 129.3, 129.9, 134.5, 167.2, 168.5,
171.9. m/z (EI+): 246 (M+, 8%), 204 (10), 91 (100). HRMS
calc. for C13H14N2O3 [M+•] 246.10044, found 246.10053.
HRMS (LSIMS with mNBA) calc. for C27H28N3O5 [MH+•
]
474.19507, found 474.20327. Crystals suitable for X-ray crystal-
lography were grown from ethyl acetate/hexanes. C27H27N3O5,
M = 473.52, T = 193 K, monoclinic, space group P21/c,
a = 10.442(5), b = 19.799(3), c = 12.147(3) Å, β = 108.83(3),
V = 2376.8(13) Å3, Z = 4, Dc = 1.323 g cm−3, specimen: yel-
low block, 0.45 × 0.45 × 0.32 mm, 4603 measured reflections,
Rint = 0.0597, r = 0.0881 for 2442 observed data ((I) > 2σ(I)),
wR = 0.2494, and GOOF = 1.065 for all data (4172).
( )-1,4-Diacetyl-3-benzyl-piperazine-2,5-dione (8)
Pyridine (0.66 mL, 8.15 mmol), acetic anhydride (0.71 mL,
7.47 mmol), and DMAP (105 mg, 0.852 mmol) were added
sequentially to a solution of 7a (1.672 g, 6.79 mmol) in dry
dichloromethane (30 mL), under an atmosphere of nitrogen.
After 18 h the reaction was quenched with saturated potassium
hydrogen sulfate solution (30 mL), the layers separated and
the aqueous phase extracted with additional dichloromethane
(2 × 25 mL). The combined organic layers were washed with
saturated potassium hydrogen sulfate (30 mL), 2 M sodium car-
bonate solution (30 mL), brine (40 mL), and then dried over
magnesium sulfate. Filtration through a plug of silica gel, eluted
with dichloromethane, yielded the title compound as a colour-
less crystalline solid (1.559 g, 95% – based on recovered starting
material), mp 83–85◦C (lit.[18] 84–85◦C). Unreacted starting
material was recovered by elution with ethyl acetate (0.2700 g,
1.09 mmol).
Alternately 8 could be prepared from compound 7b. Pyridine
(0.87 mL, 10.8 mmol), acetic anhydride (0.94 mL, 9.89 mmol),
and DMAP (177.6 mg, 1.45 mmol) were added sequen-
tially to ( )-1-acetyl-6-benzylpiperazine-2,5-dione (2.215 g,
8.99 mmol) in dry dichloromethane (40 mL), using the method
described above for ( )-1-acetyl-3-benzyl-piperazine-2,5-dione
7a, yielding the title product as a colourless crystalline solid
(2.258 g, Quant. – based on recovered starting material). Unre-
acted starting material was recovered by elution with ethyl
acetate (0.269 g, 1.09 mmol).
( )-(6Z)-3-Benzyl-6-(1H-indol-3-ylmethylene)piperazine-
2,5-dione (10)
Hydrazine hydrate (330 µL, 6.75 mmol) was added dropwise
to a solution ( )-t-butyl-3-[(Z)-(4-acetyl-5-benzyl-3,6-dioxo-
piperazin-2-ylidene)methyl]indole-1-carboxylate
(282 mg,
0.675 mmol) in DMF (5 mL), protected from light and stirred
for 18 h. The reaction was quenched with water (15 mL), and
the precipitated solid collected by filtration, washed with t-butyl
methyl ether and dried under high vacuum, yielding the title com-
pound as a yellow solid (141 mg, 63%), mp 278–280◦C dec.
νmax (solid/cm−1): 756, 1097, 1139, 1235, 1253, 1383, 1422,
1442, 1620, 1661, 3060, 3208. δH (d6-DMSO): 2.95 (dd, J 13.7,
5.2 Hz, 1H), 3.13 (dd, J 13.7, 4.5 Hz, 1H), 4.35 (m, 1H), 6.70
(s, 1H), 7.00–7.29 (m, 6H), 7.38 (d, J 7.9 Hz, 1H), 7.46 (d, J
7.9 Hz, 1H), 7.71 (d, J 2.5 Hz, 1H), 7.93 (s, 1H), 8.27 (s, 1H),
9.30 (s, 1H), 11.54 (s, 1H). δC (d6-DMSO): 36.4, 56.8, 107.7,
108.4, 112.4, 118.8, 120.4, 122.6, 122.8, 126.8, 127.3, 127.4,
128.7, 130.6, 136.2, 136.3, 161.1, 166.5. m/z (EI+): 331 (M+,
100), 240 (95), 212 (18), 155 (58), 130 (13), 91 (9). HRMS calc.
for C18H19N3O4 [M+•] 331.11716, found 331.13176.
νmax (thin film/cm−1): 704, 743, 965, 1040, 1138, 1206,
1218, 1368, 1695, 1712, 1735, 2938, 3008. δH (CDCl3): 2.44
(d, J 19.0 Hz, 1H), 2.55 (s, 3H), 2.57 (s, 3H), 3.20 (dd, J 14.0,
5.5 Hz, 1H), 3.35 (dd, J 14.0, 4.4 Hz, 1H), 4.48 (d, J 19.0 Hz,
1H), 5.43 (dd, J 5.5, 4.4 Hz, 1H), 7.02–7.08 (m, 2H), 7.22–7.32
(m, 3H). δC (CDCl3): 27.2, 27.5, 39.0, 46.3, 59.4, 128.5, 129.4,
( )-t-Butyl 3-[(Z)-(4-Acetyl-5-benzyl-3,6-dioxo-piperazin-
2-ylidene)methyl]-4-nitroindole-1-carboxylate (11)
To solution of ( )-1,4-diacetyl-3-benzylpiperazine-2,5-dione
(829 mg, 2.87 mmol) and 4-nitroindole-3-carboxaldehyde

RESEARCH FRONT
818
P. P. Molesworth et al.
(874 mg, 3.01 mmol) in dry DMF (25 mL), under an atmosphere
of nitrogen, was added cesium carbonate (1.18 g, 3.65 mmol).
After protection from light and stirring for 18 h, the reaction
was quenched with water (20 mL). The precipitated solid was
collected by filtration and washed with t-butyl methyl ether,
yielding the title compound as a yellow solid (919 mg, 61%),
mp 191–192◦C dec. νmax (thin film/cm−1): 748, 1094, 1150,
1227, 1355, 1370, 1441, 1524, 1676, 1705, 1758, 2980, 3201.
δH (CDCl3): 1.76 (m, 9H), 2.66 (s, 3H), 3.27 (ad, J 4.0 Hz, 1H),
5.35 (at, J 4.2 Hz, 1H), 6.76 (s, 1H), 7.05–7.19 (m, 2H), 7.20–
7.31 (m, 3H), 7.46 (t, J 8.2 Hz, 1H), 7.52 (s, 1H), 7.56 (bs, 1H),
7.98 (d, J 8.0 Hz, 1H), 8.57 (d, J 7.9 Hz, 1H). δC (CDCl3): 27.4,
28.3, 39.3, 58.1, 86.6, 113.4, 120.6, 121.3, 121.9, 125.4, 126.0,
127.9, 128.0, 128.8, 128.9, 130.7, 134.5, 138.0, 142.5, 148.7,
160.0, 166.1, 173.2. m/z (EI+): 518 (M+, <1%), 460 (12), 418
(40), 285 (100), 158 (28), 91 (29).
(104 mg, 0.206 mmol) in DMF (5 mL), and stirred for 24 h
protected from light. After quenching with water, the resul-
tant precipitate was collected by filtration, washed with t-butyl
methyl ether, and dried under high vacuum, yielding the title
compound as an orange solid (38 mg, 46%), mp 210–212◦C
dec. νmax (solid/cm−1): 691, 730, 795, 975, 1119, 1252, 1286,
1323, 1377, 1424, 1516, 1615, 1675, 2923, 3027, 3183. δH (d6-
DMSO): 2.64 (s, 3H), 2.90 (s, 3H), 3.05 (dd, J 13.4, 5.1 Hz,
1H), 3.18 (dd, J 13.4, 5.2 Hz, 1H), 4.43 (at, J 5.3 Hz, 1H), 6.87
(s, 1H), 7.06 (s, 1H), 7.11–7.32 (m, 6H), 7.82–7.85 (m, 2H),
12.22 (bs, 1H). δC (d6-DMSO): 33.2, 34.7, 37.8, 64.1, 107.9,
114.3, 118.3, 118.5, 119.5, 121.7, 127.6, 128.8, 129.1, 130.4,
132.0, 136.4, 139.1, 142.3, 161.6, 166.9. m/z (EI+): 404 (M+,
42%), 313 (100), 285 (20), 158 (33), 130 (22). HRMS calc. for
C22H20N4O4 [M+•] 404.14846, found 404.14861.
( )-(6Z)-3-Benzyl-6-[(4-nitro-1H-indol-
3-yl)methylene]piperazine-2,5-dione (13)
( )-(6Z)-3-Benzyl-1,4-dimethyl-6-[(4-nitro-1H-indol-
3-yl)methylene]piperazine-2,5-dione (12)
Hydrazine hydrate (200 µL, 4.16 mmol) was added dropwise to
a solution of 10 (215.9 mg, 0.416 mmol) in DMF (3 mL) and
stirred for 18 h protected from light. The reaction was quenched
with water (15 mL), the precipitated solid collected by filtra-
tion, washed with t-butyl methyl ether and dried under high
vacuum, yielding the title compound as a red solid (131 mg,
84%), mp 285–287◦C dec. νmax (solid/cm−1): 759, 806, 981,
1136, 1209, 1317, 1359, 1435, 1510, 1618, 1659, 3033, 3281.
δH (d6-DMSO): 2.98 (dd, J 13.8, 4.8 Hz, 1H) 3.14 (dd, J 13.5,
3.6 Hz, 1H), 4.30 (m, 1H), 6.61 (s, 1H), 7.03–7.36 (m, 6H),
7.53 (s, 1H), 7.79 (m, 2H), 8.29 (s, 1H), 9.53 (s, 1H), 12.19
(s, 1H). δC (d6-DMSO): 40.1, 56.8, 107.0, 109.4, 118.0, 118.6,
119.0, 121.4, 124.7, 127.3, 128.7, 130.7, 131.6, 136.3, 139.2,
142.6, 160.7, 166.4. m/z (EI+): 376 (M+, 100%), 330 (51), 285
Hydrazine hydrate (62 µL, 1.27 mmol) was added dropwise
to a solution of t-butyl ( )-3-[(Z)-(4-acetyl-5-benzyl-3,6-
dioxo-piperazin-2-ylidene)methyl]-4-nitroindole-1-carboxylate
(657 mg, 1.27 mmol) in DMF (5 mL), protected from light and
stirred for 18 h. The reaction was quenched with water (15 mL),
and the precipitated solid collected by filtration, washed with
t-butyl methyl ether and dried under high vacuum to yield ( )-t-
butyl 3-[(Z)-(5-benzyl-3,6-dioxo-piperazin-2-ylidene)methyl]-
4-nitroindole-1-carboxylate as yellow powder (577 mg, 95%),
mp 187–189◦C dec. Insolubility in common NMR solvents pre-
vented full characterization, the compound was used without
further purification. νmax (solid/cm−1): 697, 743, 790, 1091,
1154, 1278, 1434, 1526, 1634, 1676, 1743, 3069, 3187. m/z
(EI+): 476 (M+, <1%), 376 (100), 330 (54), 285 (78), 158
(48). HRMS calc. for C25H24N4O6 [M+•] 476.16958, found
476.16886.
Iodomethane (1.54 mL, 10.9 mmol) was added to a suspen-
sion of potassium carbonate (1.716 g, 12.4 mmol) and ( )-t-
butyl 3-[(Z)-(5-benzyl-3,6-dioxo-piperazin-2-ylidene)methyl]-
4-nitroindole-1-carboxylate (519 mg, 1.09 mmol) in dry DMF
(15 mL), under an atmosphere of nitrogen. After stirring for 72 h
the reaction was quenched with water (15 mL) and extracted
with ethyl acetate (3 × 15 mL). The combined organic layers
were washed with water (3 × 10 mL), and brine (2 × 25 mL),
dried over magnesium sulfate, filtered and the solvent removed
under reduced pressure. The residue was purified by col-
umn chromatography (60% ethyl acetate/hexanes), yielding
( )-t-butyl 3-[(Z)-(5-benzyl-1,4-dimethyl-3,6-dioxo-piperazin-
2-ylidene)methyl]-4-nitroindole-1-carboxylate as a crystalline
yellow solid (435 mg, 79%), mp 130–132◦C. νmax (thin
film/cm−1): 733, 919, 1101, 1258, 1285, 1315, 1352, 1433,
1522, 1635, 1682, 1743, 2934. δH (CDCl3): 1.71 (s, 9H), 2.76 (s,
3H), 3.05 (s, 3H), 3.20 (d, J 4.7 Hz, 2H), 4.32 (t, J 4.7 Hz, 1H),
6.76 (d, J 1.5 Hz, 1H), 7.04–7.30 (m, 6H), 7.37 (at, J 8.2 Hz, 1H),
7.86 (d, J 8.1 Hz, 1H), 8.45 (d, J 8.4 Hz, 1H). δC (CDCl3): 28.3,
33.4, 34.8, 37.8, 64.6, 86.1, 112.5, 113.1, 120.1, 120.9, 124.5,
127.7, 129.0, 129.2, 130.1, 130.3, 135.2, 137.1, 142.9, 148.7,
160.9, 166.6. m/z (EI+): 504 (M+, <1%), 404 (40), 313 (100),
158 (30). HRMS calc. for C27H28N4O6 [M+•] 504.20088, found
504.19859.
(78), 158 (61), 91 (91). HRMS calc. for C20H16N4O4 [M+•
376.11716, found 376.11747.
]
Methyl (2S)-2-[[2-(t-Butoxycarbonylamino)acetyl]amino]-
3-phenyl-propanoate (N-Boc-Gly-Phe-OMe) (15)
Triethylamine (5.70 mL, 40.9 mmol), then EDAC (7.32 g,
37.6 mmol) were added to a solution of l-phenylalanine methyl
ester (14) (5.62 g, 31.4 mmol) and N-Boc-glycine (6.0887 g,
34.5 mmol) in dry dichloromethane (100 mL), under an atmo-
sphere of nitrogen.The reaction was stirred for 18 h, diluted with
dichloromethane (50 mL), and the organic phase washed with
0.5 M potassium hydrogen sulfate solution (2 × 75 mL) and sat-
urated sodium carbonate solution (1 × 75 mL).The organic layer
was dried with magnesium sulfate and filtered through a plug
of silica gel (eluting with 60% ethyl acetate/hexanes), and the
solvent removed under reduced pressure, to give the title com-
pound as a viscous oil (10.15 g, 96%). δH (CDCl3): 1.40 (s, 9H),
2.95–3.15 (m, 2H), 3.64 (s, 3H), 3.67–3.77 (m, 2H), 4.84 (aq,
J 6.8 Hz, 1H), 5.41 (at, J 5.4 Hz, 1H), 6.85 (bd, J 7.4 Hz, 1H),
7.02–7.09 (m, 2H), 7.14–7.26 (m, 3H). δC (CDCl3): 28.4, 38.1,
44.2, 52.5, 53.3, 80.2, 127.3, 128.7, 129.4, 135.9, 156.2, 169.6,
172.0. m/z (EI+): 336 (M+, <1%), 280 (8), 203 (12), 162 (100),
120 (38). HRMS calc. for C17H24N2O5 [M+•] 336.16852, found
336.16799.
(3S)-3-Benzylpiperazine-2,5-dione (16)
Six batches (2 mL each) of N-Boc-Gly-Phe-OMe (4.67 g,
13.9 mmol), dissolved in ethanol (18.0 mL), were diluted with
water (2 mL) and heated to 160◦C for 1 h in a sealed tube using a
microwave reactor (CEM Discover microwave reactor). The six
Hydrazine hydrate (50 µL, 1.03 mmol) was added to a
solution of ( )-t-butyl 3-[(Z)-(5-benzyl-1,4-dimethyl-3,6-
dioxo-piperazin-2-ylidene)methyl]-4-nitroindole-1-carboxylate

RESEARCH FRONT
Synthesis and Phytotoxicity of Structural Analogues of Thaxtomin Natural Products
819
batches were combined and cooled on ice, the resulting precipi-
tate collected by filtration and dried under high vacuum, yielding
the title compound as a colourless solid (2.07 g, 73%), mp 256–
267◦C (lit.[19] 270◦C). [α]D21 = +48.4 (c 1.0 in DMSO) lit.[19]
+60 (c 0.15, DMSO). νmax (solid/cm−1): 701, 851, 1335, 1462,
1670, 2887, 3050, 3196. δH (d6-DMSO): 2.74 (d, J 17.4 Hz,
1H), 2.86 (dd, J 13.5, 4.9 Hz, 1H), 3.08 (dd, J 13.5, 4.5 Hz, 1H),
3.34 (dd, J 17.4, 2.9 Hz, 1H), 4.05 (m, 1H), 7.10–7.21 (m, 2H),
7.22–7.32 (m, 3H), 7.88 (bs, 1H), 8.14 (s, 1H). δC (d6-DMSO):
49.0, 60.9, 132.1, 133.5, 135.4, 141.4, 171.0, 172.5. m/z (EI+):
until TLC showed only one product (5 days), the reac-
tion was quenched with water (50 mL) and extracted with
ethyl acetate (3 × 25 mL). The combined organic layers were
washed with water (3 × 25 mL) and brine (2 × 50 mL), dried
over magnesium sulfate, filtered, and the solvent removed
under reduced pressure. The residue was purified by col-
umn chromatography (60% ethyl acetate/hexanes) yielding
t-butyl3-[(5S)-(Z)-(5-benzyl-1,4-dimethyl-3,6-dioxo-piperazin-
2-ylidene)methyl]-4-nitroindole-1-carboxylate as crystalline
yellow solid (245 mg, 70%). The compound was identical
by spectroscopy to the racemic material (11), [α]2D1 = −2.24
(dichloromethane).
204 (M+, 18%), 91 (100). HRMS calc. for C11H12N2O2 [M+•
204.08988, found 204.08959.
]
t-Butyl 3-[(3S)-(Z)-(5-benzyl-1,4-dimethyl-3,6-dioxo-piper-
azin-2-ylidene)methyl]-4-nitroindole-1-carboxylate (245 mg,
0.487 mmol) was reacted with hydrazine hydrate (120 µL,
2.43 mmol) in DMF (5 mL), using the method described for
the racemic compound above, yielding the title compound as
an orange solid (160 mg, 81%).The compound was identical by
spectroscopy to the racemic material (12), [α]2D1 = −23.1 (c 2.2
in DMSO).
(3S)-1,4-Diacetyl-3-benzyl-piperazine-2,5-dione ((+)-8)
Triethylamine (2.92 mL, 20.9 mmol), acetic anhydride (1.83 mL,
19.3 mmol), and DMAP (63.0 mg, 0.276 mmol) were added
sequentially to a suspension of (3S)-3-benzylpiperazine-2,5-
dione (0.6571 g, 3.22 mmol) in dry dichloromethane (30 mL)
and refluxed for 6 h. The reaction mixture was diluted with
dichloromethane (20 mL), and washed with saturated potas-
sium hydrogen sulfate solution (2 × 30 mL) and 2 M sodium
carbonate solution (1 × 30 mL). The organic layer was dried
over magnesium sulfate, filtered through a plug of silica gel
(eluting with dichloromethane), and the solvent removed under
reduced pressure, yielding the title compound as a crystalline
colourless solid (0.660 g, 71%). The compound was identical by
spectroscopy to the racemic material (7), [α]2D1 = +19.2 (c 1.4,
MeOH) lit.[19] +7.8 (c 0.52, MeOH).
(3S)-(6Z)-3-Benzyl-6-[(4-nitro-1H-indol-3-
yl)methylene]piperazine-2,5-dione ((3S)-13)
t-Butyl
3-[(3S)-(Z)-(4-acetyl-5-benzyl-3,6-dioxo-piperazin-
(195 mg,
2-ylidene)methyl]-4-nitroindole-1-carboxylate
0.376 mmol) was reacted with hydrazine hydrate (185 µL,
3.76 mmol) in DMF (3 mL), using the method described for the
racemic compound above, yielding (3S)-(6Z)-3-benzyl-6-[(4-
nitro-1H-indol-3-yl)methylene]piperazine-2,5-dione as a deep
red solid (128 mg, 91%). The compound was identical by
spectroscopy to the racemic material (13).
t-Butyl 3-[(3S)-(Z)-(4-Acetyl-5-benzyl-3,6-dioxo-piperazin-
2-ylidene)methyl]-4-nitroindole-1-carboxylate ((3S)-11)
(3S)-1,4-Diacetyl-3-benzyl-piperazine-2,5-dione
(629 mg,
Compound Toxicity Screening
2.18 mmol) was reacted with N-Boc-4-nitroindole-3-carbo-
xaldehyde (0.6568 g, 2.29 mmol) and cesium carbonate (899 mg,
2.76 mmol) in DMF (15 mL), using the method described
for the racemic compound above, yielding the title com-
pound as an off-yellow solid (738 mg, 65%). The com-
pound was identical by spectroscopy to the racemic material
(11). Crystals suitable for X-ray crystallography were grown
from CDCl3. C27H26N4O7, M = 518.52, T = 100 K, mono-
clinic, space group P21/n, a = 13.3800(11), b = 8.4840(8),
c = 22.9030(14) Å, β = 105.4840(10), V = 2505.5(3) Å3, Z = 4,
Dc = 1.375 g cm−3, specimen: yellow block, 0.15 × 0.15 ×
0.05 mm, 25381 measured reflections, Rint = 0.0481, r = 0.0384
for 3785 observed data ((I) > 2σ(I)), wR = 0.0997, and
GOOF = 1.051 for all data (3567).
Compounds (10, 12, and 13) were assayed for phytotoxic-
ity using Arabidopsis thaliana seedlings at four concentrations
(0.1 µM, 1.0 µM, 10.0 µM, 50.0 µM). Each batch had five
test compounds which were trialled at four concentrations
(0.1 µM, 1.0 µM, 10.0 µM, 50.0 µM), against untreated con-
trols containing the same volume of DMSO. For comparison,
plates containing thaxtomin A at four concentrations (0.1 µM,
0.2 µM, 1.0 µM, 2.0 µM) were also used. For each treatment
two replicated plates were prepared and 20 vernalisedArabidop-
sis thaliana seedlings per plate were pipetted directly onto the
surface of the cooled growth medium. Seeds were placed 15 mm
from the top of dish, and were individually spaced evenly across
the plate. Plates were wrapped, grouped by replicate, and trans-
ferred to a growth chamber, orientated to an upright position
of 85◦, with the seeds at the top of the plate. The seeds were
then allowed to grow along the surface of the agar for 7 days
at an ambient temperature of 22 1◦C, with a 16 h day length
(60 µmol m−2 s−1). After 7 days, the root length of 15 random
seedlings (per plate) was measured (n = 30) and compared with
the controls.
(3S)-(6Z)-3-Benzyl-1,4-dimethyl-6-[(4-nitro-1H-indol-
3-yl)methylene]piperazine-2,5-dione ((3S)-12)
t-Butyl
3-[(3S)-(Z)-(4-acetyl-5-benzyl-3,6-dioxo-piperazin-
(539 mg,
2-ylidene)methyl]-4-nitroindole-1-carboxylate
1.04 mmol) was reacted with hydrazine hydrate (51.0 µL,
1.04 mmol) in DMF (5 mL), using the method described for
the racemic compound above, yielding t-butyl 3-[(5S)-(Z)-(5-
benzyl-3,6-dioxo-piperazin-2-ylidene)methyl]-4-nitroindole-1-
carboxylate as a yellow solid (443 mg, 89%).
Iodomethane (0.440 mL, 6.93 mmol) was added to a suspen-
sion of potassium carbonate (1.014 g, 7.33 mmol) and t-butyl 3-
[(5S)-(Z)-(5-benzyl-3,6-dioxo-piperazin-2-ylidene)methyl]-4-
nitroindole-1-carboxylate (332 mg, 0.693 mmol) in dry DMF
(15 mL), under an atmosphere of nitrogen. After stirring
Compound Toxicity Range Experiments
Compounds (12 and 13) were tested to obtain IC50 values
in three batches using Arabidopsis thaliana seedlings. The
two compounds were trialled at nine concentrations (0.01 µM,
0.025 µM, 0.050 µM, 0.10 µM, 0.2 µM, 0.5 µM, 1.0 µM,
2.0 µM, 5.0 µM), in comparison to untreated controls containing
an equivalent volume of DMSO. For comparison, plates contain-
ing thaxtomin A at seven concentrations (0.01 µM, 0.025 µM,

RESEARCH FRONT
820
P. P. Molesworth et al.
0.050 µM, 0.10 µM, 0.2 µM, 0.5 µM, 1.0 µM) were also run.
For each treatment four replicated plates were prepared. Vernal-
ized Arabidopsis thaliana seedlings (20 per plate) were pipetted
directly onto the surface of the cooled growth medium. Seeds
were placed 15 mm from the top of dish, and were evenly spaced
across the plate. Plates were wrapped, randomized, and trans-
ferred to the growth chamber, orientated to an upright position
of 85◦, with the seeds at the top of the plate. The seeds were then
allowed to grow along the surface of the agar for 7 days (unless
otherwise stated) at an ambient temperature of 22 1◦C, with a
16 h day length (60 µmol m−2 s−1). After 7 days, the root length
of 15 random seedlings (per plate) was measured (n = 60 in total)
and compared with the controls.
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[5] I. Duval, V. Brochu, M. Simard, C. Beaulieu, N. Beaudoin, Planta
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[8] A. L. Johnson, T. Janosik, J. Bergman, Arkivoc 2002, viii, 57.
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[11] G. Bringmann, S. Tasler, H. Endress, K. Peters, E. Peters, Synthesis
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Accessory Publication
General experimental information and NMR spectra for new
compounds are available on the Journal’s website.
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Acknowledgements
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P.P.M. is thankful to the University ofTasmania and the School of Chemistry
for support through aThomas Crawford Scholarship.The authors thank Greg
Luckman for the isolation of thaxtomin A and Jamie Davies for assistance
in obtaining the photographs. This research was undertaken on the MX1
beamline at the Australian Synchrotron, Victoria, Australia.
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