Synthesis of d,l-amino acid derivatives bearing a thiol at the β-position and their enzymatic optical resolution

Article abstract of DOI:10.1016/j.tetlet.2015.10.015

Amino acids bearing a thiol group at the β-position are useful for native chemical ligation. Phenylalanine and tyrosine derivatives bearing a thiol group at the β-position were synthesized. Racemic phenylalanine and tyrosine were selected as starting materials and were introduced a bromo atom at the β-position by photoreaction. Subsequent substitution reaction of the bromo atom with p-methoxybenzylmercaptan yielded the corresponding amino acids bearing a thiol group at the β-position. Enzymatic optical resolution using l-aminoacylase and subsequent chemical conversion gave the corresponding optically pure l- and d-phenylalanine and tyrosine derivatives bearing a thiol group at the β-position.

Full text of DOI:10.1016/j.tetlet.2015.10.015

Tetrahedron Letters 56 (2015) 6565–6568
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of D,L-amino acid derivatives bearing a thiol
at the b-position and their enzymatic optical resolution
⇑
Yasuhito Morishita, Tomoka Kaino, Ryo Okamoto, Masayuki Izumi, Yasuhiro Kajihara
Department of Chemistry, Graduate School of Science, Osaka University, 1-1, Machikaneyama, Toyonaka, Osaka 560-0043 Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2015
Revised 30 September 2015
Accepted 5 October 2015
Available online 9 October 2015
Amino acids bearing a thiol group at the b-position are useful for native chemical ligation. Phenylalanine
and tyrosine derivatives bearing a thiol group at the b-position were synthesized. Racemic phenylalanine
and tyrosine were selected as starting materials and were introduced a bromo atom at the b-position by
photoreaction. Subsequent substitution reaction of the bromo atom with p-methoxybenzylmercaptan
yielded the corresponding amino acids bearing a thiol group at the b-position. Enzymatic optical
resolution using
pure - and -phenylalanine and tyrosine derivatives bearing a thiol group at the b-position.
Ó 2015 Elsevier Ltd. All rights reserved.
L-aminoacylase and subsequent chemical conversion gave the corresponding optically
Keywords:
L
D
Protein chemical synthesis
Native chemical ligation
Desulfurization
Amino acid
Ever since native chemical ligation (NCL) was found in 1994,
chemical syntheses of protein have gained momentum. This reac-
tion has enabled us to easily couple a peptide-a-thioester with
showed suitable reactivity in NCL and desulfurization with reduc-
tive conditions such as radical initiator (VA044), tert-butylthiol,
and (tris(2-carboxyethyl)phosphine) (TCEP).
another peptide bearing a cysteine at the N-terminus in a neutral
solution. NCL utilizes a thiol exchange reaction and the subse-
quent S-to-N acyl migration results in a native amide bond. This
powerful peptide coupling reaction allows us to synthesize not
only the native proteins but also post-translationally modified pro-
teins such as glycoproteins.
However, mild reaction conditions were selected for the
syntheses of these amino acid derivatives to avoid undesired
racemization and these strategies resulted in long synthetic steps.
These synthetic steps would hinder the preparation of correspond-
ing amino acid derivatives bearing a thiol group in sufficient
amounts for chemical protein synthesis.
1
NCL requires a cysteine residue at the ligation site and therefore
cannot be applied for the synthesis of some proteins that have no
cysteine or unsuitable number of cysteine residues in the target
polypeptide chains. In general, a number of suitable cysteine
residues are essential, as chemical synthesis of protein requires
repetitive NCL reactions employing 20–30 amino acid residue pep-
tides to construct a target polypeptide chain.
In addition to the chemical syntheses of proteins and glycopro-
teins, recently chemical synthesis of D-protein using D-amino acids
has also been demonstrated in order to examine racemic protein
crystallization and quasi-racemic glycoprotein crystallization.1
These emerging protein crystallization methodologies require both
4,15
corresponding
chemical synthetic strategy of these derivatives requires
-amino acids bearing a thiol at the b-position.
This Letter describes efficient syntheses of phenylalanine and
tyrosine bearing a thiol group at the b-position in both - and
-forms using a single synthetic procedure for the syntheses of
- and -proteins which combine the NCL and desulfurization
D-
and
L
-proteins and therefore the practical
D- and
In order to overcome these drawbacks, a recent strategy for pro-
tein synthesis employed a unique amino acid bearing a thiol group
at the b-position for NCL as a cysteine surrogate. The thiol group
was removed by desulfurization reaction resulting in the formation
L
D
L
2
of a native amino acid at the ligation site after NCL. Using this con-
D
L
cept, syntheses of amino acids bearing a thiol at the b-position or
reaction strategies.
c-position that correspond to phenylalanine, valine, leucine, lysine,
In order to synthesize corresponding amino acids bearing a thiol
proline, glutamine, threonine, asparagines, aspartic acid, trypto-
group at the b-position in both
thetic strategy using racemic amino acid as a starting material and
optical resolution employing - and -aminoacylases. Because
aminoacylase is known to be a cheap enzyme that hydrolyzes an
-amide group, this enzyme is suitable for organic synthesis and
D- and L-forms, we designed a syn-
phan, and arginine have been reported recently.3
–13
These amino
acid derivatives incorporated at the N-terminal of peptide indeed
D
L
a
⇑
Corresponding author. Tel.: +81 60 6850 5380.
E-mail address: kajihara@chem.sci.osaka-u.ac.jp (Y. Kajihara).
http://dx.doi.org/10.1016/j.tetlet.2015.10.015
040-4039/Ó 2015 Elsevier Ltd. All rights reserved.
0

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Y. Morishita et al. / Tetrahedron Letters 56 (2015) 6565–6568
Figure 1. Synthesis of N-t-Boc-DL-phenylalanine and tyrosine derivatives bearing a thiol group at the b-position. (a) SOCl
rt, (c) Boc O, 4-dimethylaminopyridine, MeCN, rt, (d) NBS, h (200 W), CCl , (e) p-methoxybenzylmercaptan, DBU, DMF, rt, (f) TFA, DCM, rt, (g) Ac
NaOH aq, rt, THF, (i) -aminoacylase, sodium phosphate buffer (20 mM, pH 8.0), 37 °C, (j) Boc O, THF, rt, (k) 2 M HCl aq, 100 °C, (l) Boc O, Na CO
3
2
, MeOH, reflux, (b) Boc
2
O, Na
O, pyridine, DCM, rt, (h) 1 M
, THF, rt.
2 3 2
CO , (THF/H O = 1:1),
2
m
4
2
L
2
2
2
there were indeed many reports employing this enzyme for optical
resolution.
for 30 min under the reflux condition gave the corresponding
brominated aromatic amino acids 5 and 6 quantitatively and these
compounds were used in the next step without further purifica-
tion. Diastereomer ratio (RR+SS/RS+SR) was estimated to be
1
6,17
A racemic mixture of commercially available phenylalanine 1
3
and tyrosine 2 was converted into b-bromo derivatives 5 and 6,
1
respectively, by photoreaction, followed by a substitution reaction
with alkylthiol. Aromatic amino acids 1 and 2 were dissolved in
ꢀ1/1 based on the integrals of corresponding H NMR signals.
Subsequent substitution reaction with p-methoxybenzylmercap-
tan yielded 7 and 8 in 74% and 37%, respectively, after silica gel
column chromatography. Diastereomer ratios of isolated 7 and 8
4
CCl and N-bromosuccinimide (NBS) was added to this solution.
Irradiation of the reaction solution with 200 W incandescent lamp
Figure 2. Confirmation of optical resolutions of
chiral column (DAICEL, OJ-RH 4.6 Â 150 mm) (a–c, e–g). HPLC analytical conditions: isocratic water/MeCN = 70:30 containing 0.1% formic acid at a flow rate of 0.8 mL/min
and detection at 254 nm. (d) Conversion of -phenylalanine derivative 13 to N-t-Boc- -phenylalanine 22.
D- and L-phenylalanine derivatives. HPLC analysis of phenylalanine derivatives bearing a thiol group at the b-position using
L
L

Y. Morishita et al. / Tetrahedron Letters 56 (2015) 6565–6568
6567
Figure 3. Confirmation of optical resolutions of tyrosine derivatives. (a–d) HPLC analysis of tyrosine derivatives bearing a thiol group at the b-position using chiral column
(
(
2
DAICEL, OJ-RH 4.6 Â 150 mm). HPLC analytical conditions: isocratic water/MeCN = 75:25 containing 0.1% formic acid at a flow rate of 0.8 mL/min and detection at 254 nm.
e) Synthesis of dipeptide by t-Boc-solid phase peptide synthesis and desulfurization. 1H NMR spectra of (f) authentic
-Tyr- -Leu, (g) authentic -Tyr- -Leu, and (h) compound
5. Asterisk ⁄ indicates water signal.
D
L
L
L
were estimated to be ꢀ4/6 and ꢀ1/4, respectively, although we did
not determine which is RR+SS or RS+SR. In this reaction we found
dehydroamino acid derivative as a byproduct. Formation of
dehydroamino acid derivative may indicate involvement of the
Michael addition reaction when racemic amino acids 7 and 8 bear-
ing a p-methoxybenzylthio group at the b-position are formed,
however we excluded such a possibility because treatment of
dehydroamino acid derivatives with p-methoxybenzylmercaptan
under the same condition did not give 7 and 8 (data not shown).
Therefore we speculated that this substitution reaction proceeds
concentration, we could not obtain H-
H- -Tyr-OH derivative 18 in moderate yields.
We thus changed the strategy to employ the chemical
conversion of -acetamide derivatives 15 and 16 into correspond-
ing -amino derivatives 17 and 18 by acid hydrolysis after com-
plete consumption of -amino acid derivative in a mixture of
racemic phenylalanine derivative 9 and tyrosine derivative 10 by
-aminoacylase. This chemical hydrolysis was performed with
2 M HCl solution at 100 °C followed by protection of amino group
with t-Boc group was performed to give Boc- -Phe-OH derivative
19 and Boc- -Tyr-OH derivative 20 in 26% and 22% yields from
racemic mixtures 9 and 10, respectively.
Because we obtained presumably - and
tives by the optical resolution based on the
egy and the chemical conversion strategy, respectively, we then
analyzed the optical purity of presumably individual - and
-phenylalanine (Fig. 2) and tyrosine (Fig. 3) derivatives bearing a
p-methoxylbenzylthio group at the b-position by a chiral column,
however this analysis did not give suitable results. Figure 2b and c
shows the isolated enantiomeric isomers of phenylalanine deriva-
tives 19 and 13, respectively, and Figure 2a shows the HPLC profile
of an intentional mixture of both phenylalanine derivatives. As
shown in Figure 2b and c, both HPLC analyses unfortunately
showed only one peak each. As individual enantiomeric amino acid
derivatives 13 and 19 have two chiral centers corresponding to the
D-Phe-OH derivative 17 and
D
D
D
L
L
D
via both S
N
2 and S
N
1 type mechanisms.
D
For subsequent optical resolution, corresponding N-di
t-Boc-phenylalanine derivative 7 and tyrosine derivative 8 were
converted into acetamide derivatives 9 and 10 through deprotec-
tion of N-di-t-Boc group followed by acetamidation in 57% and
L
D
-amino acid deriva-
L-aminoacylase strat-
5
2% yields (recrystallized as diastereomer mixtures) respectively.
Enzymatic deacetylation with -aminoacylase was then examined.
Enzymatic optical resolution toward phenylalanine derivative 9
with Aspergillus melleus -aminoacylase [EC3.5.1.14] was first
D
L
L
L
examined. This reaction was monitored by reverse phase HPLC
and this enzymatic reaction smoothly gave product 11 in reason-
able ca. 50% yield (quantitative based on
not shown). The same -aminoacylase also converted tyrosine
derivative 10 to amino derivative 12 in ca. 50% yield (quantitative
based on -substrate). The resultant 11 and 12 were used in the
next step without further purification. Amino derivatives 11 and
2 were converted into N-t-Boc-b-(p-methoxybenzylthio)- -Phe-
OH 13 and N-t-Boc-b-(p-methoxybenzylthio)- -Tyr-OH 14 with
Boc O that are suitable monomers for the t-Boc solid phase peptide
synthesis.
In terms of the optical resolution with Escherichia coli
cylase [EC3.5.1.81], each racemic mixture of phenylalanine deriva-
tive 9 or tyrosine derivative 10 was treated with -aminoacylase,
however these -aminoacylase reactions did not give products in
L-substrate, HPLC data
L
L
a- and b-positions, we thus expected to observe two peaks in both
1
L
Figure 2b and c.
L
As we could not evaluate the absolute configuration at the
2
a
-carbon using chiral columns, we instead examined the desulfur-
ization at the b-position to convert it into amino acid bearing a
chiral center at the only -carbon. Then we successfully evaluated
optical purity of individual - and -phenylalanine derivatives.
D-aminoa-
a
L
D
D
Deprotection of 13 gave thiol 21 and subsequent radical desulfur-
ization reaction with VA-044, tert-butylthiol and TCEP gave 22
(Fig. 2d). The resultant compound 22 was compared with authentic
D
suitable yields. Despite our extensive optimization of reaction
conditions with regard to temperature, pH and substrate
N-t-Boc-L- and D-phenylalanine. Figure 2e shows the purity of the

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Y. Morishita et al. / Tetrahedron Letters 56 (2015) 6565–6568
2
5
product 22 ([
column and Figure 2f shows an HPLC profile of a mixture of 22 with
authentic N-t-Boc- -phenylalanine (commercially available sam-
À12.4, c 1.0, MeOH). Figure 2g shows a mixture of 22
with authentic N-t-Boc- -phenylalanine (commercially available
+13.3, c 1.0, MeOH). Figure 2e–g clearly indicates
the product of desulfurization reaction toward 13 to be homoge-
neous N-t-Boc- -phenylalanine. This result also indicated that
optical resolution with -aminoacylase recognizing the -carbon
a
]
D
+15.1, c 0.6, MeOH) by HPLC using a chiral
we assume that both diastereomers would be usable in NCL even
though there may be some differences in reaction velocities.4
D
In conclusion, we successfully synthesized N-t-Boc-
D- and
2
5
ple: [
a
]
D
L
-phenylalanine and new N-t-Boc- - and -tyrosine bearing a
D
L
L
p-methoxybenzylthio group at the b-position in 10 steps and 11
2
5
sample: [
a
]
D
steps, respectively. Although 10-step conversion for the synthesis
3
of the
L
-phenylalanine derivative is the same compared to that
L
of a previous report, the conversion yield elevated from 9% to
16% (total yield of the previous synthesis in 11 steps). More impor-
tantly, our synthetic strategy makes it unnecessary to consider
L
a
was successfully performed. Figure 2a–c show that the separation
of 13 and 19 resulted from the difference of chirality at the
racemization. We can easily separate corresponding
acids, both of which are useful building blocks for racemic protein
crystallization, via enzymatic optical resolution using -aminoacy-
D- and L-amino
a-carbon of the phenylalanine derivative. Through these chemical
conversions and analyses using a chiral column, we could clearly
L
confirm that we obtained the desired N-t-Boc-phenylalanine
lase and the chemical conversion strategy. Research to obtain other
amino acids bearing a thiol group at the b-position is currently in
progress.
bearing a thiol group at the b-position in both
D
- and
L
-forms in
sufficient yield.
We also analyzed
L-tyrosine derivative 14 and D-tyrosine
derivative 20 (Fig. 1) using a chiral column, however, the chiral
column could not separate these derivatives (Fig. 3a). We already
knew that racemic acetamide derivative 10 (Fig. 1), a precursor
for the L-aminoacylase reaction, showed a well-separated HPLC
profile on the chiral column (data not shown). We therefore
Acknowledgments
This work was supported in part by Grant-in-Aid for Scientific
Research from the Ministry of Education, Sports, Science and
Technology of Japan (No. 26248040 and 23245037).
converted 14, which was prepared by the treatment of 10 with
L
-aminoacylase and subsequent protection with Boc
acetamide derivative 23 (Fig. 3b: synthetic scheme is not shown).
The resulting derivative was compared with N-Ac- -tyrosine
derivative 16 that had remained after the treatment of racemic
mixture 10 by -aminoacylase reaction (Fig. 3c).
2
O, into the
Supplementary data
D
Supplementary data (experimental procedures, and spectral/
characterization data) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.10.
L
As shown in Figure 3b–d, although both N-Ac-tyrosine deriva-
tives 16 and 23 were well separated by the chiral column, we could
not confirm whether this separation resulted from the chirality of
0
15.
References and notes
the
using N-t-Boc-
Boc-solid phase peptide synthetic conditions to confirm the
absolute configuration of the -carbon. After purification, synthetic
a-carbon or b-carbon. We therefore prepared a dipeptide by
L
-tyrosine derivative 14 and -leucine under the
L
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a
dipeptide 24 was treated with TCEP, tert-butylthiol and VA-044 for
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authentic L-Tyr-L-Leu and D-Tyr-L-Leu using NMR. As shown in
Figure 3f–h, individual NMR spectra clearly indicated that the
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at the b-position were
As shown in Figures 2 and 3, we confirmed the absolute config-
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bearing a thiol at the b-position in both - and -forms. These data
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1
5