C3 and 2D C3 Marfey's Methods for Amino Acid Analysis in Natural Products

Article abstract of DOI:10.1021/acs.jnatprod.5b01125

We validate the improved resolution and sensitivity of the C₃ Marfey's method, including an ability to resolve all Ile isomers, against an array of amino acids commonly encountered in natural products and by comparison to an existing Marfey's m

Full text of DOI:10.1021/acs.jnatprod.5b01125

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Article
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C and 2D C Marfey’s Methods for Amino Acid Analysis in Natural
3
3
Products
†
†
Soumini Vijayasarathy, Pritesh Prasad, Leith J. Fremlin, Ranjala Ratnayake, Angela A. Salim,
Zeinab Khalil, and Robert J. Capon*
Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia
*
S Supporting Information
ABSTRACT: We validate the improved resolution and sensitivity
of the C Marfey’s method, including an ability to resolve all Ile
3
isomers, against an array of amino acids commonly encountered in
natural products and by comparison to an existing Marfey’s
method. We also describe an innovative 2D C Marfey’s method
3
as an analytical approach for determining the regiochemistry of
enantiomeric amino acid residues in natural products. The C and
3
2
D C Marfey’s methods represent valuable tools for probing and
3
defining the stereocomplexity of hydrolytically accessible amino
acid residues in natural products.
1
irst described in 1984, Marfey’s method has emerged as a
Notwithstanding the widespread use of Marfey’s, advanced
Marfey’s, and C Marfey’s methods, an enduring shortcoming of
F
popular and effective procedure for determining the
absolute configurations of hydrolytically accessible amino acid
residues in natural products (typically but not exclusively
peptides). This method employs four steps: (i) the natural
product to be analyzed is subjected to acid hydrolysis (6 M HCl)
to release amino acid residues, (ii) the hydrolysate is derivatized
under alkaline conditions (1 M NaHCO ) with Nα-(2,4-dinitro-
-fluorophenyl)-L-alaninamide (L-FDAA) or the enantiomer
Nα-(2,4-dinitro-5-fluorophenyl)-D-alaninamide (D-FDAA), (iii)
the neutralized derivatized analyte is subjected to HPLC-UV
18
all these methods has been the inability to differentiate L-Ile from
3
,6
L-allo-Ile, and D-Ile from D-allo-Ile. With Ile commonly
occurring in natural products, this limitation is problematic.
For example, although the Marfey’s method was correctly
acknowledged as incapable of resolving Ile and allo-Ile
9
diastereomers in the tetrapeptide asperterrestide A, this did
3
5
not preclude it being used to assign L-Ile residues in
1
0
11
12
microcystilide A, dichotomins J and K, euryjanicin A,
C
13
14
15
and D, and E−G, aeruginopeptins, micropeptins 88-N and
16 17−19 20
(
340 nm) analysis, and (iv) HPLC retention times of derivatized
88-Y, kailuins A−F,
talaromins A and B, callyaerins A, B,
23 24
2
1,22
amino acids in the analyte are compared with those of derivatized
L and D amino acid standards. Key advantages of this method
include improved detection sensitivity compared to under-
ivatized amino acids, the use of readily available HPLC
technology, and the commercial availability of both enantiomers
of the chiral derivatizing agent (CDA) FDAA. The latter
represents a significant but often underappreciated benefit. For
example, where a D-amino acid standard is not available, the D-
FDAA-L-amino acid derivative is a chromatographically (i.e.,
analytically) equivalent surrogate for the L-FDAA-D-amino acid
derivative. Consequently, Marfey’s analyses can be successfully
performed with L-FDAA and D-FDAA derivatives of L-amino acid
standards, without the need to source the frequently less
accessible D-amino acid standards.
D, and J−L,
trichoderins A and A1, falcitidin,
26
25
mullinamide A, and stylissatin A and D-Ile residues in
27
28
29
anthranicine, PF1171C, PF1171F, and PF1171G. Signifi-
cantly these assignments focused on distinguishing L-Ile from D-
Ile, without reference to allo isomers.
Alert to the allo option, other researchers employed
adaptations of Marfey’s method, including (i) chiral-phase
3
0
HPLC to identify L-Ile residues in companeramides A and B,
31
32
L-allo-Ile in desomatides C and D, myriastramides A−C, and
33
34
hirsutellide A, D-allo-Ile in aspergillicins A, C, and D and
34
35
hytramycin I, and L-allo-Ile and D-allo-Ile in aspergillicin E;
ii) multiple (tandem) HPLC columns to identify D-allo-Ile in
(
36
37
nostophicin and yaku’amides A and B and L-allo-Ile in
38,39
lagunamides A−C;
and (iii) normal-phase (cellulose) HPLC
40
to identify L-Ile and D-allo-Ile in teixobactin, a very promising
new class of antibiotic recently reported from an uncultured
bacterium. Despite case-specific successes, none of these HPLC
adaptations were systematically validated against all standard
amino acids, and none have been adopted as a generic solution to
Over the last three decades several efforts have been made to
2
−6
improve Marfey’s method.
One prominent example, the
advanced Marfey’s method, introduced the CDA Nα-(2,4-
dinitro-5-fluorophenyl)-L-leucinylamide (L-FDLA), its racemate
(
L/D-FDLA), and a modified HPLC protocol supportive of
7,8
ESIMS detection (Table 1). Other adaptations incorporate ad
hoc C18 HPLC protocols, herein collectively referred to as C18
Marfey’s methods (Table 1).
Received: December 18, 2015
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Article
Table 1. Summary of Prominent Marfey’s Methods
d
HPLC method
CDA
column (temp)
solvent elution
detection
UV (340 nm)
a
Marfey’s
L-FDAA or D-
FDAA
reversed-phase (room linear gradient 10% to 50% MeCN in 50 mM triethyl ammonium phosphate at pH 3.0
temp)
a
Advanced Marfey’s
L-FDLA and D/L-
FDLA)
C18 (40 °C)
1 mL/min, 45 min linear gradient elution from 25% to 65% MeCN/H O, with an isocratic TFA UV (340 nm) +
2
b
(0.01 M) modifier
ESIMS
,
ac
C18 Marfey’s
L-FDAA or D-
FDAA
C18 (room temp)
1 mL/min, 45 min linear gradient elution from 15% to 45% MeCN in 0.1 M NH OAc/TFA buffer UV (340 nm)
4
at pH 3.0
C Marfey’s
3
L-FDAA or D-
FDAA
C (50 °C)
3
1 mL/min, 55 min linear gradient elution from 15% to 60% MeOH/H O with a 5% isocratic 1% UV (340 nm) +
2
formic acid in MeCN modifier
ESIMS
a
b
c
Cannot resolve D/L-Ile from D/L-allo-Ile. Flow rate and gradient profiles are optimized on a case-by-case basis. This is an indicative HPLC
4,5 d
method, as many variants have been reported.
In all these methods, while absolute retention times of individual amino acids will vary between
laboratories (e.g., HPLC configurations and columns), relative retention times are preserved. Retention times can be especially sensitive to pH and
isocratic solvent modifier concentrations (note: as % TFA and formic acid can vary with storage, these should be prepared fresh).
Figure 1. Derivatized amino acid standards analyzed by the C Marfey’s method. Black horizontal line: Difference in retention times between L-FDAA-L-
3
AA/D-FDAA-D-AA (earlier t ) and D-FDAA-L-AA/L-FDAA-D-AA (later t ). Vertical cyan line: Residual Marfey’s reagent. Red circle: Enantiomers are
R
R
not resolved. Blue circle: Enantiomers are resolved by diderivatized analogues. *Not resolved.
the Ile challenge. Yet other researchers addressed the assignment
Ile configuration and biological activity. The examples cited
of Ile configurations by modifying the CDA, for example, using
above illustrate the need for a more effective analytical
methodology, capable of rapid and cost-effective assignment of
configurations to all amino acid residues encountered in natural
products, inclusive of Ile.
2
,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate to iden-
41 42
tify L-Ile in sungsanpin and L-Ile and D-Ile in surugamides, or
resorted to chemical degradation, identifying N-Me-L-Ile and L-
43
Ile residues in epoxomicin, or total synthesis, identifying L-allo-
Another enduring challenge in the structure elucidation of
peptidic natural products has been assigning the regiochemistry
of enantiomeric amino acid residues. For example, while the
regiochemistry of L- and D-Ala residues in the cyclohexadep-
sipeptide paecilodepsipeptide A was inferred (unconvincingly)
4
4
45
Ile in desotamide and D-allo-Ile and L-Ile in aspergillicin F.
The aspergillicins are an especially noteworthy case, with the L-
Ile-containing aspergillicin F being a suppressor of the innate
immune system and its L-allo-Ile stereoisomer aspergillicin E
exhibiting no suppressor activity, affirming a correlation between
46
from NOESY correlations, the regiochemistry of L- and D-Asn
B
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Figure 2. Derivatized amino acid standards analyzed by the C Marfey’s method. Black horizontal line: Difference in retention times between L-FDAA-
18
L-AA/D-FDAA-D-AA (earlier t ) and D-FDAA-L-AA/L-FDAA-D-AA (later t ). Vertical cyan line: Residual Marfey’s reagent. Red circle: Enantiomers are
R
R
not resolved. Blue circle: Enantiomers are resolved by diderivatized analogues. *Not resolved; however, enantiomer resolution can be achieved with a
modified C18 Marfey’s method [see Experimental Section].
4
7
in mojavensin A and L- and D-Leu in the cyclohexapeptide
sufficient to solve the regiochemical challenge posed by
enantiomeric amino acid residues, and other approaches are
labor intensive and costly in material and time.
3
1
48
desotamides B−D and desotamide G were asserted without
comment, and the regiochemistry of L- and D-Leu residues in the
lipopeptide hallobacillin was left unassigned. As evidence of the
effort required to secure unambiguous assignments, the
regiochemistry of L- and D-Leu in the cyclohexapeptide
49
Collectively, the examples cited above illustrate the need for
improved analytical methodology capable of assigning both
amino acid configurations and regiochemistry to enantiomeric
amino acid residues in natural products. In responding to this
need we set out to refine existing Marfey’s methods, retaining the
well-regarded L-FDAA and D-FDAA and focusing our efforts on
developing a more effective, reproducible, and readily accessible
achiral HPLC method with enhanced levels of resolution. Aware
that natural products chemists deal with very small quantities of
hard-won metabolites, we were also alert to the need for
enhancing sensitivity. Illustrative of this latter point, the majority
of Marfey’s analyses cited above involved the acid hydrolysis of
44
desotamide A relied on the total synthesis of multiple isomers,
while the regiochemistry of L- and D-Ile in the cyclooctapeptide
surugamides A−E relied on partial hydrolysis to tri- and
dipeptide fragments, which were in turn identified by a
combination of Marfey’s analyses and HPLC comparison to six
synthetic dipeptides. The regiochemistry of L- and D-N-Me-Ala
in companeramide B was determined by a combination of partial
acid hydrolysis, followed by HPLC isolation of a diagnostic
tetrapeptide fragment, and an advanced Marfey’s analysis. This
regiochemical challenge is also well exemplified by kahalalide F,
first isolated in 1993 as an exceptionally potent cytotoxic cyclic
peptide from the sarcoglossan mollusk Elysia rufescens and its
dietary green alga (Bryopsis sp.). The structure assigned to
kahalalide F on the basis of NMR, acid hydrolysis, and chiral-
phase GC-MS analysis was inclusive of five Val residues,
42
30
0
.5−5.0 mg of natural product, a level of consumption that is
both excessive and in many instances impractical. Our aspiration
was to develop a highly sensitive analytical Marfey’s method
capable of rapid and unambiguous assignment of configuration
and enantiomer regiochemistry, across all commonly encoun-
tered amino acids.
50
including both L- and D-Val. Although a 1999 chemical
degradation proposed the regiochemistry of L- and D-Val (and all
RESULTS AND DISCUSSION
■
51
52
other amino acid) residues, subsequent chemical synthesis
After extensive trials employing multiple achiral HPLC columns
and numerous elution profiles (see Supporting Information), we
settled on a C HPLC column held at 50 °C, with a H O/MeOH
5
3
and degradation studies led to a revision of regiochemistry.
Significantly, these latter studies confirmed that the unnatural D/
L-Val regioisomer was significantly less cytotoxic than the natural
regioisomer, confirming the ecological/pharmacological impor-
tance of accurately determining regiochemistry. Significantly, to
date no implementation of Marfey’s method alone has proved
3
2
gradient elution modified by isocratic 1% formic acid in MeCN,
taking advantage of both UV (340 nm) and ESI(±)MS
detection, the latter further enhanced by single ion extraction
filters. To validate the resolution of this C Marfey’s method, we
3
C
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Figure 3. Published natural products incorporating amino acid residues identified by the C Marfey’s method.
3
Figure 4. HPLC-DAD-MS chromatograms extracted from a C Marfey’s method analysis of 50 μg of desotamide A (7) derivatized with D-FDAA: (a)
3
UV (340 nm), where * = residual D-FDAA; (b) SIE (m/z 328) for D-FDAA-Gly (shaded); (c) SIE (m/z 386) for D-FDAA-L-Asp (shaded); (d) SIE (m/z
=
384) for D-FDAA-D-Leu, D-FDAA-L-allo-Ile, and D-FDAA-L-Leu (shaded); (e) SIE (m/z = 457) for D-FDAA-L-Trp (shaded). Note for (b)−(e) FDAA
derivatives of authentic amino acid standards are also displayed (broken line).
applied it to all standard and several nonstandard (N-
methylated) amino acids, with comparisons to a typical C18
Marfey’s method (Figures 1 and 2, respectively). This analysis
confirmed that the C Marfey’s method was capable of resolving
3
D
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all amino acids, including Ile stereoisomers (Figure S5).
have been relatively insensitive (i.e., operating on a 4−15 mg
30,51,53
Moreover, in the C Marfey’s method the residual Marfey’s
scale acid hydrolysis)
and often relied on the total
3
reagent did not obscure any target amino acids, unlike the C18
Marfey’s method, where residual reagent can mask L-FDAA-L-
Tyr, L-FDAA-L-N-Me-Ala, D-FDAA-L-Pro, and D-FDAA-L-Ala.
synthesis and chromatographic and/or spectroscopic compar-
42
ison to multiple putative hydrolysis products. Significantly,
none of these “partial hydrolysis” approaches have gained
acceptance as a generally applicable solution. By contrast, the 2D
During this development phase we applied the C Marfey’s
3
method to assign amino acid configurations in the lipodepsipep-
C Marfey’s method operates at an analytical scale without
recourse to chemical synthesis, as illustrated by its assignment of
the L- and D-Leu regiochemistry in 7 (Figure 5). A short-duration
3
54
tide acremolides [e.g., D-Ile and L-Pro in acremolide C (1)],
cyclopentapeptide cotteslosins [e.g., L-allo-Ile, L-Val, L-Tyr, N-
55
Me-L-Tyr, and L-Pro in cotteslosin B (2)] and macrolide
polyketide nocardiopsins [e.g., L-pipecolic acid in nocardiopsin A
5
6,57
(3)],
as well as the anticancer N-formyl amino-salicylamide
58
neoantimycins [e.g., L-Thr in neoantimycin G (4)], the
antimalarial glycol-hexadepsipeptide-polyketide mollemycin A
5
9
(
5), and the antibacterial cyclohexapeptide desotamides and
wollamides [e.g., L-Trp, L-Leu, D-Leu, L-allo-Ile, L-Asn, and D-Orn
60
in wollamide A (6)] (Figure 3). Significantly, all these studies
were performed at an analytical scale (50−100 μg) well below
that routinely reported in the literature for Marfey’s, advanced
Marfey’s, and C Marfey’s methods. In addition to providing a
18
comprehensive documentation and validation of our C Marfey’s
3
method against a suite of amino acids commonly encountered in
natural products (Figure 1), we take this opportunity to describe
a new Marfey’s derivatization, the 2D C Marfey’s method, which
3
can assign regiochemistry to enantiomeric amino acid residues.
The utility of the C and 2D C Marfey’s methods is illustrated on
3
3
desotamide A (7), as summarized below.
In a 1997 report describing the DeSota Falls soil Streptomyces
cyclic hexapeptide desotamide A (7), the assignment of amino
acid configuration and the regiochemistry of L- and D-Ile residues
44
relied on the total synthesis of multiple isomers. In 2014 the
rediscovery of 7, together with the new congeners desotamides E
and F and wollamides A and B, from an Australian desert soil
Streptomyces provided an opportunity to showcase the C3
Marfey’s method as a means to rapidly identify the configurations
of all amino acid residues. This process involved the acid
hydrolysis of 7 (50 μg) followed by derivatization with D-FDAA
and HPLC-DAD-MS analysis to generate the UV (340 nm)
chromatogram illustrated in Figure 4a. While the UV detection
approach employed by established Marfey’s methods is an
improvement over underivatized amino acids, it nevertheless
suffers from several limitations. These include the relative
instability of Trp to acid hydrolysis (i.e., leading to reduced
sensitivity) and the masking effect of residual Marfey’s reagent
and near-identical retention times for several derivatized amino
acids (i.e., leading to reduced resolution). In an effort to
Figure 5. HPLC-DAD-MS chromatograms extracted from a 2D C3
Marfey’s method analysis of 150 μg of desotamide A (7) derivatized with
D-FDAA: (a) UV (340 nm), where * = residual D-FDAA; (b) SIE (m/z
4
97) revealing (i) as D-FDAA-D-Leu-L-allo-Ile (green peak) and (iii) as
D-FDAA-L-Leu-D-Leu (red peak). Inset corresponds to the SIE (m/z
84) for D-FDAA-derivatized D-allo-Ile, L-allo-Ile, D-Leu, and L-Leu
60
3
standards (broken line) and the D-FDAA-derivatized acid hydrolysate of
peak (i) (shaded). (c) SIE (m/z 570) revealing (ii) as D-FDAA-L-Trp-L-
Leu (blue peak). Inset corresponds to the SIE (m/z 384) of the
standards as per (b) and the D-FDAA-derivatized acid hydrolysate of
peak (ii) (shaded).
partial hydrolysis of 7 (150 μg) generated peptide fragments that
were derivatized with D-FDAA and analyzed by HPLC-DAD-
MS. The UV (340 nm) chromatogram (Figure 5a) revealed an
array of partial hydrolysis peptide fragments, with SIE chromato-
grams at m/z 497 (Figure 5b) and 570 (Figure 5c) selecting for
the diagnostic dipeptides D-FDAA-L/D-Leu-L-allo-Ile and/or D-
FDAA-L/D-Leu-D/L-Leu (i and iii) and D-FDAA-L-Trp-L/D-Leu
(ii), respectively. The well-resolved peak (i) was purified by
analytical HPLC and subjected (without further character-
ization) to acid hydrolysis and derivatization with D-FDAA. A
overcome these limitations, the C Marfey’s method employs
3
ESIMS detection and single-ion extraction (SIE). For example, in
the case of 7, individual SIE chromatograms at m/z 328 (Figure
4b), 386 (Figure 4c), 384 (Figure 4d), and 457 (Figure 4e)
readily distinguished the constituent amino acids by MW (i.e.,
providing enhanced sensitivity), with comparison to the
retention times of L- and D-FDAA derivatives of L amino acid
standards confirming these as Gly, L-Asp, D-Leu, L-allo-Ile, L-Leu,
and L-Trp (i.e., providing enhanced resolution). Note, although
Asn converts to Asp under acid hydrolysis, this does not preclude
assignment of its absolute configuration.
subsequent C Marfey’s analysis (Figure 5b inset) confirmed the
3
presence of both D-Leu and L-allo-Ile, unambiguously establish-
ing the regiochemistry of the D- and L-Leu residues in 7. As
further evidence of this assignment, a comparable sequence of
analyses carried out on peak (ii) confirmed that L-Leu (Figure 5c
inset) was adjacent to L-Trp. While the broad utility of the 2D C3
Marfey’s method is conditional on the complexity of the partial
hydrolysate of the target natural product, it is nevertheless a
valuable analytical tool that should find general application in the
field of natural products chemistry.
With an effective C Marfey’s method in hand, we set out to
3
develop a 2D Marfey’s (partial hydrolysis followed by total
hydrolysis) adaptation as a tool for assigning the regiochemistry
of enantiomeric amino acid residues in peptidic natural products,
which we refer to as the 2D C Marfey’s method. While partial
3
hydrolysis Marfey’s approaches have been used in the past, these
E
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Journal of Natural Products
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In summary, this report provides a detailed account of the C3
Marfey’s method, documenting enhanced resolution and
sensitivity in assigning configurations to hydrolytically accessible
amino acid residues commonly encountered in natural products.
This report also describes an innovative adaptation, the 2D C3
Marfey’s method, capable of determining the regiochemistry of
enantiomeric amino acid residues in peptidic natural products,
on an analytical scale and without recourse to chemical synthesis.
These two methods represent valuable new analytical tools that
can be used to probe the stereocomplexity of amino acid residues
in natural products.
and D-FDAA as detailed in the Supporting Information. Aliquots (5 μL)
of each analyte were injected into an Agilent Zorbax Eclipse XDB-C18
HPLC column, 5 μm, 150 × 4.6 mm, 25 °C (rt), with a 1 mL/min, 45
min linear gradient elution from 15% to 45% MeCN in an NH OAc/
4
TFA buffer adjusted to pH 3.0. The amino acid content in analytes was
assessed by UV (340 nm), with comparison to authentic standards.
A Modified C18 Marfey’s Method (N-Me-Ala). Natural product
acid hydrolysis and the synthesis of FDAA derivatives of the acid
hydrolysate and of amino acid standards were performed as indicated
above for the C Marfey’s method. Aliquots (5 μL) of each analyte were
18
injected into an Agilent Zorbax SB-C18 HPLC column, 5 μm, 150 × 4.6
mm, 50 °C, with a 1 mL/min, 35 min isocratic elution of 23% MeOH/
H O with a 5% isocratic modifier of 1% formic acid in MeCN. These
2
condition resolved L-FDAA-N-Me-L-Ala (t = 23.3 min) from D-FDAA-
R
EXPERIMENTAL SECTION
N-Me-L-Ala (t = 24.2 min).
■
R
General Experimental Procedures. Nα-(2,4-Dinitro-5-fluoro-
phenyl)-L-alaninamide (L-FDAA, synonym 1-fluoro-2,4-dinitrophenyl-
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b01125.
■
5
-L-alanine amide) and Nα-(2,4-dinitro-5-fluorophenyl)-D-alaninamide
*
S
(
D-FDAA, synonym 1-fluoro-2,4-dinitrophenyl-5-D-alanine amide)
were purchased from NovaBiochem. Amino acids and standards were
purchased from NovaBiochem, BAChem Biosciences, Sigma, Fluka, or
Merck. HPLC-grade MeCN and MeOH were degassed and filtered
through a 0.45 μm polytetrafluoroethylene (PTFE) membrane prior to
use. Water for HPLC was filtered through an ELGA PurelabUltra system
prior to degassing and further filtration through a 0.45 μm hydrophilic
polypropylene membrane. All other chemicals and solvents were of
analytical grade.
HPLC-DAD-ESIMS. Analyses were performed on an Agilent 1100
series separations module equipped with a quaternary pump, vacuum
degasser, well plate autosampler with thermostat control, thermostated
column compartment, diode array detector detection (DAD) system
with analytical flow cell, and a single quadrupole electrospray ionization
mass detector operating in both positive and negative ionization modes,
over m/z 100 to 1000.
HPLC chromatograms, development of the C Marfey’s
method, and experimental details for 2D C Marfey’s
method (PDF)
3
3
AUTHOR INFORMATION
Corresponding Author
Tel: +61 7 3346 2979. Fax: +61 7 3346 2090. E-mail: r.capon@
uq.edu.au.
Author Contributions
S. Vijayasarathy and P. Prasad contributed equally.
Notes
■
*
†
C3 Marfey’s Method (Desotamide A). A sample of 7 (50 μg) in 6
M HCl (100 μL) was heated to 100 °C in a sealed vial for 8−12 h, after
The authors declare no competing financial interest.
which the hydrolysate was concentrated to dryness at 40 °C under a
stream of dry N . The hydrolysate was then treated with 1 M NaHCO3
2
ACKNOWLEDGMENTS
(
20 μL) and D-FDAA (1% solution in acetone, 40 μL) at 40 °C for 1 h,
■
after which the reaction was neutralized with 1 M HCl (20 μL) and
filtered (0.45 μm PTFE) prior to HPLC-DAD-ESIMS analysis. An
This research was funded in part by the Institute for Molecular
Bioscience, The University of Queensland, and the Australian
Research Council. P.P., S.V., R.R., and L.J.F. acknowledge UQ for
Postgraduate Research Scholarships.
aliquot (10 μL) of the analyte was injected into an Agilent Zorbax SB-C3
column, 5 μm, 150 × 4.6 mm, 50 °C, with a 1 mL/min, 55 min linear
gradient elution from 15% to 60% MeOH/H O with a 5% isocratic
2
modifier of 1% formic acid in MeCN. The analyte amino acid content
was assessed by UV (340 nm) and ESI(±)MS monitoring, supported by
SIE, with comparison to authentic standards.
REFERENCES
■
(
(
1) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
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2
D C Marfey’s Method (Desotamide A). A sample of 7 (150 μg)
3
7
(
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2
(
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̈
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