Modular control ofl-tryptophan isotopic substitutionviaan efficient biosynthetic cascade

Article abstract of DOI:10.1039/d0ob00868k

Isotopologs are powerful tools for investigating biological systems. We report a biosynthetic-cascade synthesis of Trp isotopologs starting from indole, glycine, and formaldehyde using the enzymesl-threonine aldolase and an engineered β-subunit of tryptop

Full text of DOI:10.1039/d0ob00868k

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Oligonucleotide modifi cations enhance probe stability for
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DOI: 10.1039/D0OB00868K
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Modular Control of L-Tryptophan Isotopic Substitution via an
Efficient Biosynthetic Cascade
a
a
a
a
Clayton M. Thompson, Allwin D. McDonald , Hanming Yang , Silvia Cavagnero *, and Andrew R.
Buller *
Received 00th January 20xx,
Accepted 00th January 20xx
a
DOI: 10.1039/x0xx00000x
Isotopologs are powerful tools for investigating biological systems.
We report a biosynthetic-cascade synthesis of Trp isotopologs
starting from indole, glycine, and formaldehyde using the enzymes
L-threonine aldolase and an engineered β-subunit of tryptophan
synthase. This modular route to Trp isotopologs is simple and
inexpensive, enabling facile access to these compounds.
Isotopically substituted amino acids are valuable tools for the
1
mechanistic and structural analysis of biological systems.
Among the standard amino acids, L-tryptophan (Trp) is the most
structurally complex and serves as the precursor to diverse
2
natural products and clinically used compounds. Selective
isotopic substitution of Trp is often essential for determining
the metabolic fate of individual atoms and the kinetic
3
,4
properties of enzymes that manipulate Trp. Further, the low
prevalence of Trp in proteins makes this amino acid attractive
5
,6
for selective substitution methodologies for protein NMR. In
each of these cases, however, access to the requisite Trp
isotopolog is a central hurdle.
While there are diverse synthetic methods that can produce
amino acids, making isotopically substitution compounds
comes with unique challenges. For instance, synthetic routes
should be planned starting from the cheapest possible
isotopically substituted precursor. The typical concerns over
catalyst loading and atom economy are secondary to yield with
respect to the expensive, isotopically labelled or substituted
reagents. Traditional synthetic routes that can access
isotopically substituted Trp include the asymmetric Strecker
synthesis (Scheme 1) and reductive amination. These
approaches, however, often require isotopically enriched
starting materials that are not commercially available or are
prohibitively expensive. Additionally, most applications of
Scheme 1. Synthetic and proposed routes to L-tryptophan
(Trp). Kp = K , potassium phosphate buffer.
i
x 4
D3-xPO
amino acids like Trp benefit from high enantiopurity and chiral
small molecule catalysts that are often difficult to obtain.
Enzymatic Trp synthesis is an attractive alternative with the
potential for both high selectivity and low environmental
impact. For example, previous studies on enzyme mechanism
7
8
2
utilized a (2- H)Trp that was prepared biocatalytically from
2
indole and S-methyl cysteine in D O using the enzyme
tryptophanase (Enzyme Commission (EC) 4.1.99.1, Scheme 1).
Although this procedure was successful, it afforded only 40%
9
2
2
yield after a 2-day reaction. The synthesis of a [3,3- H ]Trp
presented a more substantial challenge. To make this
isotopolog, methionine γ-lyase (EC 4.4.1.11) was used to
catalyze the exchange of the three protons on the 2- and 3-
a. Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave.
Madison, WI. 53706.
†
Footnotes relating to the title and/or authors should appear here.
positions of S-ethyl-L-cysteine with D
in a 47% yield, followed by reaction with indole and
2
O solvent over three days
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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1
0
tryptophanase. Methionine γ-lyase accomplishes this labeling
through desaturation at C3 during the course of its reversible
reaction. Unfortunately, the resulting Trp was composed of a
mixture of mono, di-, and tri-deutero isotopologs. Biocatalytic
View Article Online
DOI: 10.1039/D0OB00868K
1
3
approaches have also been used to prepare various C-
substituted Trp by the action of tryptophan synthase (EC
.2.1.20) on the correspondingly substituted C-Ser. This C-
1
3
11
13
4
Ser was prepared from isotopically substituted glycine (Gly) and
formaldehyde using the enzyme Ser hydroxymethyltransferase
(
SHMT, EC 2.1.2.1). This method is particularly attractive, as Gly
isotopologs are relatively cheap. However, long reactions times
>80 h) were required, and contamination with unreacted Gly
necessitated tert-butyloxycarbonyl (Boc)-protection followed
(
1
3
by column chromatography to isolate the C-Ser, reducing
yields of the combined process to < 30%. Here, we propose an
affordable and efficient method to generate Trp starting from
Gly, formaldehyde, and indole in a one-pot, two-step enzymatic
cascade reaction that leads to the facile synthesis of
enantiopure Trp isotopologs.
We began by considering the synthesis of Trp from Ser and
indole. Previous efforts used wild-type tryptophan synthase
from Salmonella typhimurium, which is a multi-enzyme complex
that is tedious to prepare for synthetic applications.1 The β-
subunit of the complex (TrpB) is responsible for the pyridoxal
phosphate (PLP)-dependent condensation of Ser and indole.
Recently, the TrpB protein from the hyperthermophilic archeon
Pyrococcus furiosus (PfTrpB) was engineered for activity outside
2
1
Figure 1. H-NMR spectra of synthesized Trp isotopologs.
1
2
H spectra of unlabeled commercial Trp (top), (2- H)Trp
middle), and (2- C, 2, 3, 3- H )Trp (bottom). See
3
supporting information for more detail.
13
2
(
1
3,14
of its native complex.
Detailed studies have subsequently
need for intermediate isolation (Scheme 1, Route 2). We chose
the C-terminally His-tagged LTA from Thermatoga maritima
TmLTA) due to its high thermal stability and successful prior
applications in biocatalysis. In our hands, heterologous
overexpression of TmLTA in E. coli yielded ~1 g of protein L
culture (Figure S1). We began developing the two-enzyme
cascade on analytical scale by combining TmLTA and PfTrpB
shown that a stand-alone variant containing eight mutations,
PfTrpB2 , has structural and kinetic properties that are nearly
B9
(
1
5
identical to those of the parent complex. We overexpressed
1
6
PfTrpB2 in E. coli and purified the enzyme using Ni-affinity
B9
-
1
1
5
chromatography. This procedure routinely yielded > 500 mg
protein L-1 culture. This high expression level is particularly
important for practical synthetic applications. To achieve the
quantitative deuteration at the 2-position of Trp, we deployed
PfTrpB2 for the condensation of Ser and indole in deuterated
2
B9
B9
buffer (Scheme 1, Route 1). This biocatalytic reaction produced
2
(
2- H)Trp in 89% isolated yield (18.2 mg, Figure 1, Table S1).
Given the high yield and excellent properties (expression,
speed, thermal stability, etc.) of the catalyst, this synthesis of
2
(
2- H)Trp represents a significant improvement over previous
routes. However, synthesis of more sophisticated Trp
1
3
isotopologs, including the desirable 2- C substitution, is
hampered by the significant cost of the corresponding Ser
isotopolog.
Previous efforts to synthesize isotopologs of Ser from Gly
utilized the PLP-dependent SHMT enzyme, which uses the
expensive tetrahydrofolate cofactor to deliver formaldehyde.
This route was further stymied by the need for [Boc]-
Figure 2. Optimization of TmLTA-PfTrpB2B9 cascade
1
1
derivatization and isolation of the Ser isotopolog. We
hypothesized that Ser could be produced from formaldehyde
and Gly using an L-threonine aldolase (LTA, EC 4.1.2.5). We
reasoned that even though the synthetic direction of the LTA
reaction is unfavorable (equilibrium lies towards Gly and
reaction. Reference reaction conditions are 5 mM Gly, 5
mM indole, 5 mM formaldehyde, 0.005 mM LTA and
2B9
PfTrpB , and 0.02 mM PLP and modified as indicated.
Reactions were run in quadruplicates and analyzed by
UPLC-MS. Uncertainties in percent yield relative to glycine
are reported as ± standard error.
2
B9
formaldehyde), the coupled transformation with PfTrpB will
provide a strong thermodynamic driving force and obviate the
2
| J. Name., 2012, 00, 1-3
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and explored the effect of variable substrate and catalyst Marfey’s reagent analog and subsequent UPLC-MS analysis (SI
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1
7
DOI: 10.1039/D0OB00868K
loading. Given that our primary goal was not to explore Fig. S2). Hence, the optimized reaction conditions led to
1
3
2
mechanistic differences among substrate isotopologs, we formation of isotopically pure (2- C, 2, 3, 3- H
3
)Trp in 60%
found it operationally simplest to compare cascade efficiencies isolated yield (4.8 mg).
under different conditions by evaluating endpoint-product
formation. We observed that excess formaldehyde did not its potential utility, we carried out 2- C NMR longitudinal (T
result in increases in product formation (Figure 2). Excess and transverse (T ) relaxation measurements on unlabeled Trp
To further characterize the above Trp isotopolog and probe
1
3
1
)
2
1
3
2
formaldehyde increased formation of a species whose mass and (2- C, 2, 3, 3- H
(
3
)Trp (Figure 3). Theory predicts that
and T , consistent
m/z = 217) is consistent with formation of an undesired β- deuterium substitution results in increased T
1
2
carboline through a spontaneous Pictet-Spengler reaction. with our experimental observations. This result likely arises
Indeed, comparing reaction with a full equivalent or only 0.75 from the decrease in the magnetic dipole-dipole interaction due
1
8
equivalents of formaldehyde gave similar amounts of product to introduction of the selective deuterium substitution. The
1
3
(
Figure 2). Increasing the concentration of indole relative to the decreased C relaxation rates can be extremely useful for
other substrates did not increase Trp formation. Reactions protein NMR analysis, which typically suffers from short T s due
proceeded to higher conversions as the substrates were diluted, to slow protein tumbling rates. In addition, the 2-C signal of
2
1
3
13 13
achieving almost quantitative conversion at 5 mM of each uniformly C-substituted Trp is split by C- C J coupling from
substrate with the ideal ratio of 1:1:1 Gly, formaldehyde, and the neighboring nuclei. In contrast, this coupling is not present
1
3
2
indole. The catalyst loadings to achieve this conversion were (2- C, 2, 3, 3- H
fairly high for enzymatic reactions, 20 µM of each enzyme, more intense, sharper 2- C resonance.
3
)Trp. The above combined factors result in a
1
3
corresponding to 0.4 mol% catalyst. In practical terms,
In conclusion, we have developed a simple and modular
however, the high catalyst loadings are compensated for by the one-pot method for the synthesis of enantiopure Trp
2
high expression of each enzyme.
isotopologs. The simpler (2- H)Trp can be accessed through a
This new biocatalytic cascade is highly modular with respect single enzymatic reaction. Independent control of substitution
1
3
to isotope incorporation (Scheme 1, Route 2). The (2- C)Trp at the 2- and 3- positions can be achieved through a dual
1
3
substitution can be incorporated from commercially available enzyme cascade, as demonstrated by the synthesis of (2- C, 2,
1
3
2
2
2B9
(
3
2- C)Gly, and (2- H) substitution (deuteration at the α- 3, 3- H )Trp. We note that each of the enzymes, PfTrpB and
position) is achieved via exchange with a deuterated solvent, as TmLTA, can be overexpressed to exceptionally high protein
shown above. Substituting at the 3-position can be controlled density in E. coli (0.5 − 1.0 g protein L- culture) facilitating
via the source of formaldehyde, which is commercially available preparative-scale reactions. We envision that this modular
as a variety of isotopologs. As an exemplar of this the new approach could be implemented with any source of selectively
cascade methodology, we employed the two-enzyme cascade or uniformly substituted glycine, formaldehyde, or indole,
1
1
3
2
3
to synthesize the complex (2- C, 2, 3, 3- H )Trp isotopolog which are all commercially available, to yield diverse
starting from the appropriate isotopically enriched precursors. isotopically-substituted Trps. The resulting products promise to
This particular Trp isotopolog was chosen because of its serve as valuable tools in mechanistic biochemical studies as
potential as a resonance-assignment aid for protein NMR well as structural-biology investigations.
spectroscopy. In addition, this compound showcases the
2
B9
modular nature of the PfTrpB -LTA cascade. In order to
characterize the extent of C incorporation at position 2 of Trp
and D incorporation at positions 2 and 3, we carried out UPLC-
1
3
Conflicts of interest
A.R.B is an inventor on a patent on the use of PfTrpB for the
synthesis of tryptophans.
2B9
1
13
MS as well as H and C NMR analysis (Figure 1, S5-S8). These
2
data show a complete absence of the 2-H in (2- H)Trp. In
1
3
addition, the 2- C signal, 2- and 3-H signals are absent from the
1
3
2
(
2- C, 2, 3, 3- H
3
)Trp. The enantiomeric excess (ee) for both Trp
Acknowledgments
isotopologs was found to be > 99% via derivatization with a
We are grateful to Jon Ellis for helpful scientific discussions and
to Aadhishre Kasat and Meghan Campbell for the synthesis of
FDVA. S.C. thanks the National Institute of Health (grant
R01GM125995) and the UW-Madison Graduate School for
funding. A.R.B. gratefully acknowledges support from the
National Institute of Health New Innovator Award
(DP2GM137417). The Bruker AVANCE 400 NMR spectrometer was
supported by NSF grant CHE-1048642, and the Bruker AVANCE 600
NMR spectrometer was supported by NIH grant S10 OD012245.
1
3
2
1 2 3
Figure 3. T and T measurements for (2- C, 2, 3, 3- H )Trp
and unlabeled commercial Trp.
References
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Figure 3. T1 and T2 measurements for (2-13C, 2, 3, 3-2H3)Trp and unlabeled commercial Trp.

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