A new pre-column derivatization for valienamine and beta-valienamine using o-phthalaldehyde to determine the epimeric purity by HPLC and application of this method to monitor enzymatic catalyzed synthesis of beta-valienamine

Article abstract of DOI:10.1080/10286020.2017.1292257

Valienamine and β-valienamine are representative C₇?N aminocyclitols with significant glycosidase inhibition activity that have been developed as important precursors of drugs for diabetes and lysosomal storage diseases, respectively. The quantitative analysis of these chiral compounds is crucial for asymmetric in vitro biosynthetic processes for converting valienone into valienamine epimers using aminotransferase. Here, we developed an efficient and sensitive method for separation and quantitative analysis of chiral valienamine using reversed-phase high-performance liquid chromatography (HPLC) through o-phthalaldehyde (OPA) pre-column derivatization of the analytes. The epimers were derivatized by OPA in borate buffer (pH 9.0) at room temperature for 30 s, separated on an Eclipse XDB-C18 (5?μm, 4.6?×?150?mm) column, eluted with 22% acetonitrile at 30?°C for 18?min, and detected by a fluorescence detector using 445?nm emission and 340?nm excitation wavelengths. The average resolution of the epimers is 3.86, and the concentration linearity is in the range of 0.02–20?μg/ml. The method proved to be effective, sensitive, and reliable with good intra- and inter-day precision and accuracy, and successfully evaluated the enantiopreference and catalytic capability of the potential aminotransferases on an unnatural prochiral substrate, facilitating the design of an asymmetric biosynthetic route for optically pure valienamine and β-valienamine.

Full text of DOI:10.1080/10286020.2017.1292257

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
A new pre-column derivatization for valienamine
and beta-valienamine using o-phthalaldehyde
to determine the epimeric purity by HPLC and
application of this method to monitor enzymatic
catalyzed synthesis of beta-valienamine
Li Cui, Xiao-Qing Guan, Zhang-Min Liu, Liu-Yin Fan, Qian Li & Yan Feng
To cite this article: Li Cui, Xiao-Qing Guan, Zhang-Min Liu, Liu-Yin Fan, Qian Li & Yan
Feng (2017) A new pre-column derivatization for valienamine and beta-valienamine using o-
phthalaldehyde to determine the epimeric purity by HPLC and application of this method to monitor
enzymatic catalyzed synthesis of beta-valienamine, Journal of Asian Natural Products Research,
19:4, 347-357, DOI: 10.1080/10286020.2017.1292257
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1292257
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Date: 07 April 2017, At: 09:49

Journal of asian natural Products research, 2017
Vol. 19, no. 4, 347–357
http://dx.doi.org/10.1080/10286020.2017.1292257
A new pre-column derivatization for valienamine and
beta-valienamine using o-phthalaldehyde to determine the
epimeric purity by HPLC and application of this method to
monitor enzymatic catalyzed synthesis of beta-valienamine
Li Cuia, Xiao-Qing Guana, Zhang-Min Liua, Liu-Yin Fana, Qian Lib and Yan Fenga
astate Key laboratory of Microbial Metabolism, school of life science & Biotechnology, and Joint international
research laboratory of Metabolic & developmental sciences, shanghai Jiao tong university, shanghai
200240, china; bschool of life science & technology, china Pharmaceutical university, nanjing 210009, china
ABSTRACT
ARTICLE HISTORY
received 8 december 2016
accepted 3 february 2017
Valienamine and β-valienamine are representative C7 N aminocyclitols
with significant glycosidase inhibition activity that have been
developed as important precursors of drugs for diabetes and
lysosomal storage diseases, respectively. The quantitative analysis of
these chiral compounds is crucial for asymmetric in vitro biosynthetic
processes for converting valienone into valienamine epimers using
aminotransferase. Here, we developed an efficient and sensitive
method for separation and quantitative analysis of chiral valienamine
using reversed-phase high-performance liquid chromatography
(HPLC) through o-phthalaldehyde (OPA) pre-column derivatization of
the analytes. The epimers were derivatized by OPA in borate buffer (pH
9.0) at room temperature for 30 s, separated on an Eclipse XDB-C18
(5 μm, 4.6 × 150 mm) column, eluted with 22% acetonitrile at 30 °C
for 18 min, and detected by a fluorescence detector using 445 nm
emission and 340 nm excitation wavelengths. The average resolution
of the epimers is 3.86, and the concentration linearity is in the range
of 0.02–20 μg/ml. The method proved to be effective, sensitive, and
reliable with good intra- and inter-day precision and accuracy, and
successfully evaluated the enantiopreference and catalytic capability
of the potential aminotransferases on an unnatural prochiral
substrate, facilitating the design of an asymmetric biosynthetic route
for optically pure valienamine and β-valienamine.
KEYWORDS
Valienamine; chiral
amine; epimer separation;
derivatization; hPlc
CONTACT Qian li
leqean@163.com ; Yan feng
yfeng2009@sjtu.edu.cn
© 2017 informa uK limited, trading as taylor & francis Group

348
L. CUI ET AL.
1. Introduction
Valienamine and β-valienamine (1-epi-valienamine) are both C7 N aminocyclitols, which
have pseudosugar structures similar to pyranoses, except for the ring oxygen being replaced
with a carbon atom. ey serve as optically building blocks for the synthesis of the inhibitors
of glycosidases [1,2]. Valienamine (Figure 1(a)) exhibits α-glycosidase inhibition activity and
has been determined to be the key moiety in the crop protectant validamycin A and the anti-
diabetic drug acarbose [3,4]. As an important pharmaceutical precursor, valienamine can
be chemically converted into valiolamine [5] and further modified to voglibose [6], which
is another popular clinical therapeutic for type II diabetes [7]. β-Valienamine (Figure 1(b))
shows promising β-glycosidase inhibitor activity for the opposite amino group stereochem-
istry [8,9]. Its two derivatives, N-octyl-β-valienamine and N-octyl-4-epi-β-valienamine,
have been proven to be promising chemical chaperone therapeutic agents for lysosomal
storage diseases, including Gaucher disease, GM1-gangliosidosis, and Morquio B disease
caused by the disorder of β-glycosidase [10,11].
e chemical synthetic routes for valienamine and β-valienamine are quite challenging
due to poor stereospecificity of chemical catalysts. e routes also suffer from multiple
steps, harsh reaction conditions, and a proclivity to generate chemical pollutants [12,13].
An asymmetric biosynthetic strategy starting from the prochiral substrate valienone and by
employing aminotransferase to produce optically pure valienamine or β-valienamine, based
on the stereoselectivity of the candidate enzyme (Figure 2), was designed as a prospective
alternative. Hence, a reliable separation and quantitative analysis method is required to
separate the valienamine and β-valienamine epimers to evaluate the stereospecificity of
the enzymatic products.
HPLC is a top choice for detection of chemicals with high precision and accuracy, and
is superior to traditional thin-layer chromatography (TLC) analysis of valienamine [14].
A pre-column derivatization HPLC analysis method for valienamine was reported previ-
ously by Shen and co-workers [15]. Valienamine was detected and quantified through a
pre-column derivatization procedure using p-nitrofluorobenzene and a UV detector, which
required a harsh derivatization protocol using high temperatures (100 °C) for 30 min, with
anhydrous samples redissolved in dimethylformamide. Kim’s group described a quantitative
analysis of valiolamine, a homologue of valienamine, derivatized with phenylisocyanate
(PHI) [16]. at process also suffered from the disadvantage of a tedious procedure wherein
the samples had to be dried and redissolved in dimethyl sulfoxide before analysis because
Figure 1. structures of (a) valienamine and (b) β-valienamine.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
349
Figure 2. asymmetric synthetic strategy for optically pure valienamine epimers by aminotransferase
from the prochiral substrate.
PHI is sensitive to water. Most recently, Wang’s group improved the method with 4-meth-
oxybenzenesulfonyl fluoride (MOBS-F) as the derivatization reagent, which can react with
valiolamine in aqueous solution [17]. However, it required a highly acidic solution as the
elution mixture, consisting of 85% phosphate buffer (pH 3.0), which is especially damaging
to the column and affects downstream mass spectrum signals. No other research on HPLC
analysis for valienamine and β-valienamine epimers has been reported to date.
In this study, a reliable, sensitive, and facile HPLC analysis procedure is developed
through pre-column derivatization by o-phthalaldehyde (OPA) and detection by a fluo-
rescence detector (FLD). It is not only suitable to evaluate the asymmetric biosynthesis of
chiral amines such as valienamine and β-valienamine, but can also conveniently connect
in series with mass spectrometry (MS) for the identification of unknown chemicals and
natural products with primary amino groups.
2. Results and discussion
2.1. Establishment of the derivatization conditions
Derivatization by chromophoric reagents is essential for the analysis of valienamine epi-
mers because of their high polarity and because the absence of chromophores or fluoro-
phores in their structures causes difficulty in both separation and detection processes. e
derivatization was designed around the amino group of the valienamine epimers. OPA
has been selected as the chiral derivatization reagent for amino acid analysis [18,19] over
other derivatization reagents as its derivatization reaction is very facile, allowing an online
process and thereby eliminating errors from manual operation and shortening the time
interval between derivatization and detection. Furthermore, the derivatization reaction only
occurs with primary amines, thereby eliminating the interference of secondary amines in
complex samples such as microbial media. Considering these advantages, OPA was suitable
for the separation and quantification of valienamine and β-valienamine by monitoring the
formation of R,S-configuration chiral amino groups on the hexatomic ring of the prochiral
substrate via the derivatization reaction shown in Figure 3.
To improve the efficiency of the derivatization for better detection sensitivity, the effects of
the derivatization temperature, buffer pH, and reaction time were investigated by an L25(56)
orthogonal experimental design with three factors and five levels at fixed concentrations of
valienamine (10 μg/ml) and OPA (0.2 M). e reaction temperature was tested from 20 to

350
L. CUI ET AL.
Figure 3. schemefortheputativereactionofthederivatizationofvalienamineandβ-valienaminewithoPa.
40 °C, buffer pH from 8 to 12, and reaction time from 30 to 150 s by the gradients shown
in Table 1.
Comparing the chromatographic peak area data, the results of the orthogonal test and
range analysis showed the following order of the relative importance of the reaction fac-
tors: pH > temperature > time. Reaction pH was the most notable factor, and the optimal
derivatization pH was 9.0. e reaction temperature and time had only a slight impact on
the derivatization process.
Using the optimal conditions determined above, the concentration of OPA was tested
from 0.01 to 0.20 M at room temperature for 30 s, and it was seen that the peak area of the
valienamine derivative products reached a plateau at 0.1 M OPA (Figure 4). Taking into
account all factors and the possible consumption of OPA by the amino donor L-glutamine in
the enzymatic asymmetric synthetic system, as well as amino acids in the microbial culture
medium, the optimal online derivatization conditions were 0.15 M OPA in a 0.40 M borate
buffer (pH 9.0) at room temperature for 30 s before separation.
2.2. Establishment of the separation and detection conditions
To obtain the most sensitive detector response, the optimal wavelength for FLD was inves-
tigated. e optimal emission wavelength was determined to be 445 nm by scanning from
280 to 600 nm with the excitation wavelength fixed at 240 nm. en, the excitation wave-
length was determined to be 340 nm by scanning from 214 to 400 nm with the emission
wavelength fixed at 450 nm. e characteristic emission wavelength was determined at
445 nm and excitation wavelength at 340 nm.
Unlike drug quality control, biosynthetic or fermentation process monitoring always
needs to be combined with mass spectrometry for active component discovery and iden-
tification. Considering that a high concentration borate buffer is especially damaging to
the column and also influences the MS signal, the mobile phase was prepared with (A)
ultra-pure water and (B) 100% acetonitrile.
e two epimers were applied to the chromatographic system in pure form to obtain
their individual elution profiles, and the effects of different experimental conditions on the
resolution of the 1:1 epimer mixture were investigated. e resolution of the valienamine

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
351
Table 1. factors of the orthogonal test for the derivatization of valienamine.
Level
Temp (°C)
pH
Time (s)
1
2
3
4
5
20
25
30
35
40
8
9
10
11
12
30
60
90
120
150
Figure 4. effect of concentration of oPa on the amount of valienamine derivatives.
epimers was observed to be more sensitive to the mobile phase gradient ratio and flow rate
than column temperature. Satisfactory separation was obtained with β-valienamine, which
shows a longer retention time than valienamine under the chosen conditions.
Volume ratios of acetonitrile in the total mobile phase from 10 to 50% were examined,
and the separation profiles were compared. e results showed that the retention time of the
derivatives decreased with increasing acetonitrile concentration. Flow rates between 0.5 to
1.0 ml/min afforded appropriate separation resolution. Flows rate greater than 1.5 ml/min
or lower than 0.4 ml/min led to overlapped peak forms. e optimal separation gradient
parameters determined for the valienamine epimers – considering both resolution and
efficiency of different experimental conditions – are given in Table 2, which shows that the
retention times of valienamine and β-valienamine were 5.1 and 5.7 min with a resolution
of 3.67 at an epimer concentration of 10 μg/ml (Figure 5(a)).
e same characteristic ion peaks, with m/z 352.1209 and 374.1027, were observed in the
mass spectra of the two epimers under Electrospray Ionization (ESI) positive scan mode,
corresponding to the two peaks of HPLC. ese matched well with the theoretical values
for [M + H]+ and [M + Na]+ ions of the valienamine and β-valienamine derivative products
(Figure 5(b)). is evidence indicated that valienamine and β-valienamine were successfully
derivatized by OPA and separated under optimal elution conditions. e entire procedure
for epimer analysis was approximately 20 min, including derivatization, separation, and
detection, which is more efficient than previous methods.

352
L. CUI ET AL.
Table 2. hPlc gradients for the separation of the epimers.
Time (min)
Solvent (%)
0
8
12
18
ultra-pure water i
acetonitrile
78
22
0
100
78
22
78
22
note: flow rate: 1.0 ml/min, column temperature 30 °c.
Figure 5. separation and identification of valienamine epimer derivatives. (a) separation of valienamine
and β-valienamine at 10 μg/ml; (b) positive esi mass spectra of the oPa-derivatized valienamine, which
has the same characteristic ion peaks as the derivatized β-valienamine.
2.3. Validation and verification
2.3.1. System suitability parameters
Valienamine and β-valienamine showed average resolution of 3.86 0.008 in 0.02–20 μg/
ml of the 1:1 mixtures under the optimized conditions, with good peak shape and high
column efficiency as expressed by the asymmetry factors and theoretical plate numbers
shown in Table 3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
353
Table 3. Validation parameters of the method.
Valienamine
β-Valienamine
asymmetry factor (as)a
resolution (r)a
0.8285 0.018
3.86 0.008
19,238
0.02–20
y = 120.42x + 0.8726
0.9994
0.8395 0.04
3.86 0.008
19,520
theoretical plate number (n)a
linear range (μg/ml)
regression equation
R2
0.02–20
y = 114.35x + 0.9533
0.9997
lod (μg/ml)
0.002
0.002
loQ (μg/ml)
0.01
1.22
1.73
102.69
0.01
1.46
1.69
98.82
intra-day precision (% r.s.d., n= 6)b
inter-day precision (% r.s.d., n=6)b
standard recovery (%, n=9)
spiked samples recovery (%, n=9)
100.43
101.09
asystem suitability parameters were evaluated at epimer concentrations of 0.02–20 μg/ml.
bintra-day and inter-day precision was calculated at an epimer concentration of 10 μg/ml.
Figure 6. chromatograms of oPa derivatives. (a) Blank reaction system; (b) blank reaction system spiked
with valienamine, and (c) spiked with β-valienamine.
2.3.2. Selectivity
An asymmetric synthetic reaction system without any enzyme was separated and detected
as a blank control. e chromatographic baseline from 3 to 12 min was stable (Figure 6(a)),
and the spiked valienamine and β-valienamine were eluted at 5.1 (Figure 6(b)) and 5.7 min
(Figure 6(c)), respectively. is indicated that the derivatives in the blank reaction system
did not interfere with the separation and detection of the valienamine epimers.
2.3.3. Linearity and sensitivity
e linearity and sensitivity of the method were evaluated under the optimal derivatization
and separation conditions, using gradual concentrations of the epimers as the horizontal
coordinate (x) and the peak area as the vertical coordinate (y) to calculate the regression
equation for the standard curves. e linearity test of the calibration range was carried
out over the range of 0.02–20 μg/ml with the standard epimers using FLD. e calibration

354
L. CUI ET AL.
curves were linear in the studied range with good correlation coefficients, and the linear
regression equations for the corresponding curves are shown in Table 3.
e limit of detection (LOD) and limit of quantification (LOQ) were defined as S/N = 3
and 10, respectively. e LOD of the epimers detected by FLD was 0.002 μg/ml for the
standard mixture solution. e LOQ was 0.01 μg/ml, which is a high sensitivity for FLD.
2.3.4. Precision and accuracy
Six replicate samples were averaged on the same day and over six continuous days to deter-
mine intra-day and inter-day precision values, respectively. e relative standard deviations
(% R.S.D.) for the intra-day precision of valienamine and β-valienamine were 1.22 and 1.46,
and for the inter-day precision, the values were 1.69 and 1.73, respectively.
e accuracy of the epimers was expressed in terms of absolute recoveries of standards
and spiked samples in the blank enzymatic reaction system. Concentrations of 0.05, 1.0, and
8.0 μg/ml were detected by FLD. Every sample was repeated three times, and the average
recoveries are shown in Table 3, reflecting the reproducibility and precision of the method.
2.4. Determination of epimers in the enzymatic reaction system
Biocatalysts have emerged as a powerful alternative to traditional chemical catalysts, espe-
cially for asymmetric synthesis [20,21]. One key requirement during process development
is the discovery of a biocatalyst with appropriate enantiopreference and enantioselectivity
[22,23]. We focused our efforts to develop a biosynthetic strategy for optically pure valien-
amine and β-valienamine through the prochiral substrate, which was deeply reliant on the
precise analysis of the epimers.
Sixteen candidate sugar aminotransferases from different bacteria [24,25] were tested
for their catalytic ability to produce chiral valienamine or β-valienamine. Mixtures of the
enzymatic reaction samples were diluted and analyzed under the optimal conditions, and
six aminotransferases from different sources were found to recognize the unnatural sub-
strate valienone and showed different characteristic enantiopreferences (Table 4). Among
these, BtrR (Figure 7(a)), Per, DesI, and WbpE demonstrated stringent stereospecificity
with >99.9% epimeric purity to produce β-valienamine, but with different catalytic abil-
ity. Other candidates had lower stereospecificity and produced an epimer mixture. For
example, ArnB (Figure 7(b)) from Escherichia coli gave a 60.1% epimeric purity for the
β-valienamine product.
e analytical method developed herein allowed us to successfully evaluate the enan-
tiopreference and catalytic capability of the candidate aminotransferases on the unnat-
ural substrate while designing the asymmetric biosynthetic route for valienamine and
β-valienamine.
Table 4. catalytic properties of candidate enzymes used to produce β-valienamine.
Enzyme
Source
Valienamine (μg/ml)
β-Valienamine (μg/ml)
Epimeric purity (%)
Btrr
Per
desi
Wbpe
ntda
arnB
Bacillus circulans
–
–
–
–
26.0
42.8
1875.7
191.6
173.2
151.3
161.7
172.0
>99.9
>99.9
>99.9
>99.9
72.3
Caulobacter crescentus
Streptomyces venezuelae
Pseudomonas aeruginosa
Bacillus subtilis
Escherichia coli
60.1

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
355
Figure 7. hPlc analysis of the enzymatic reaction under the optimal conditions. different candidate
enzymes produced (a) optically pure β-valienamine (Btrr) and (b) an epimer mixture (arnB).
3. Experimental
3.1. Chemicals and reagents
Valienamine was purchased from Carbosynth China Ltd. (Suzhou, China). Valiolamine
was from YaoXing Pharmaceutical Technology Co., Ltd. (Wuxi, China). β-valienamine
was purified through an enzymatic reaction with the structure and purity (98%) verified
by NMR. o-Phthalaldehyde (OPA) and β-mercaptoethanol (β-ME) were purchased from
Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China). Acetonitrile was from ANPEL
Laboratory Technologies Inc. (Shanghai, China), and the other chemicals were purchased
from Sigma-Aldrich (St Louis, USA). All chemicals were of analytical reagent grade, unless
stated otherwise.
3.2. Standard solution preparation
e standard solution was prepared by dissolving valienamine and β-valienamine in distilled
water to give a final concentration of 200 μg/ml. e solution was successively diluted with
distilled water to create a concentration gradient from 0.2 to 200 μg/ml.
3.3. Sample preparation for the enzymatic reaction
e enzymatic asymmetric synthesis reaction system was composed of 5 mM valienone
(prochiral substrate), 5 mM L-glutamine (amino donor), and 0.3 mM pyridoxal 5′-phos-
phate (cofactor). All the chemicals were incubated in 20 mM potassium phosphate (pH 7.5)
with the purified candidate aminotransferase at 37 °C for 3 h. e reaction was terminated
by methanol as a protein precipitation agent and centrifuged at 12,000 rpm for 10 min. e
supernatant was filtered through 0.22 μm membranes before analysis.
Spiked samples were prepared as control blanks using the protocol for making asym-
metric synthetic reaction solutions, but without the addition of enzyme.

356
L. CUI ET AL.
3.4. Chromatographic analysis
Chromatographic separation and detection was performed on an Agilent 1260 Infinity
HPLC system with a quaternary pump (G1311C), standard autosampler (G1329B), ther-
mostatted column compartment (G1316A), and fluorescence detector (G1321B). High-
resolution mass spectral analysis was performed using an Agilent 1290-MS 6230 TOF-MS
system in positive mode.
Valienamine and β-valienamine standards and the test samples were detected through
pre-column derivatization by OPA and β- mercaptoethanol in H3BO3 buffer. Separation
and quantification of the derivatives were performed on an Eclipse XDB-C18 (5 μm,
4.6 × 150 mm) column, and the samples were eluted with a gradient of acetonitrile at
30 °C and monitored by FLD.
3.5. Calibration and validation
Both the derivatization and separation conditions were optimized to determine an automatic
online procedure. e asymmetry factor (As), resolution (R) and theoretical plate number
(N) of the epimer standards were calculated to evaluate the suitability of the separation
parameters such as peak shape and inter-sample separation ability. Selectivity, linearity,
sensitivity, precision, and accuracy were investigated to validate the method.
4. Conclusions
is is the first demonstration of a separation procedure for valienamine and β-valienamine
epimers through the use of a chiral derivatization reagent. e method was shown to be
sensitive and reliable and a suitable necessary tool; it successfully helped in the construc-
tion of an asymmetric synthetic route generating valienamine and β-valienamine with
high stereospecificity. It represents a promising strategy for chemical synthesis employing
biological agents.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was financially supported by the Science and Technology Commission of Shanghai
Municipality [grant number 14JC1403500]; National Science Foundation of China [grant num-
ber 31200061]; and Research Fund for the Doctoral Program of Higher Education [grant number
13Z102090066].
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