N(?)-2-Naphthylmethoxymethyl-Protected Histidines: Scalable, Racemization-Free Building Blocks for Peptide Synthesis

Article abstract of DOI:10.1021/acs.oprd.9b00538

Histidine (His) racemizes with relative ease during peptide synthesis. One strategy to suppress this racemization is to protect the nitrogen atom of the imidazole moiety in His with a suitable protecting group. Among the numerous protecting groups that have already been tested, the p-methoxybenzyloxymethyl (PMBOM) group on the ?-nitrogen atom effectively suppresses the racemization. However, a large-scale synthesis of N(?)-PMBOM-protected derivatives has hitherto been hampered by the requirement of a freshly prepared unstable reagent. Herein we report the synthesis of N(?)-2-naphthylmethoxymethyl (NAPOM)-protected His derivatives, which can be prepared on a gram scale and do not suffer from the aforementioned instability problems. Furthermore, these NAPOM-protected His derivatives suppress the racemization in Boc- A nd Fmoc-based peptide synthesis.

Full text of DOI:10.1021/acs.oprd.9b00538

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Communication
N(#)-2-Naphthylmethoxymethyl-Protected Histidines: Scalable,
Racemization-Free Building Blocks for Peptide Synthesis
Kohei Torikai, Ryota Yanagimoto, and Louis A Watanabe
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00538 • Publication Date (Web): 19 Feb 2020
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N(π)-2-Naphthylmethoxymethyl-Protected Histidines: Scalable, Rac-
emization-Free Building Blocks for Peptide Synthesis
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,†
†
‡
Kohei Torikai,* Ryota Yanagimoto, and Louis A. Watanabe
†
Department of Chemistry, Faculty and Graduate School of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka
19-0395, Japan
8
‡
Watanabe Chemical Industries, Ltd., 2-2-5 Sakaimachi, Naka-ku, Hiroshima 730-0853, Japan
Supporting Information Placeholder
ABSTRACT: Histidine (His) racemizes with relative ease during
peptide synthesis. One strategy to suppress this racemization is the
protection of the nitrogen atom of the imidazole moiety in His with
a suitable protecting group. Among the numerous protecting groups
that have already been tested, the p-methoxybenzyloxymethyl
(PMBOM) group on the -nitrogen atom effectively suppresses the
racemization. However, a large-scale synthesis of N()-PMBOM-
protected derivatives has hitherto been hampered by the require-
ment of a freshly prepared unstable reagent. Herein, we report the
synthesis of N()-2-naphthylmethoxymethyl (NAPOM)-protected
His derivatives, which can be prepared on a gram scale and do not
suffer from the aforementioned instability problems. Furthermore,
these NAPOM-protected His derivatives suppress the racemization
in Boc- and Fmoc-based peptide synthesis.
that it was still unsatisfactory given the presence of the free -ni-
trogen atom, which is considered to act directly as a base. Therefore,
currently developed His building blocks bear protecting groups
6
7
8
such as phenacyl, allyl, benzyloxymethyl (BOM), t-butyloxyme-
9
10ab
thyl (Bum) and p-methoxybenzyloxymethyl (PMBOM) on the
5
-nitrogen atom, as these groups suppress the racemization more
efficiently. Among these, N()-fluorenylmethoxycarbonyl
(Fmoc)- and N()-PMBOM-protected His derivative 5 (Chart 1) is
particularly useful, considering that the PMBOM group can be
cleaved readily with trifluoroacetic acid (TFA) following the
Fmoc-based peptide synthesis. Despite its synthetic utility, 5 has
not found widespread applications, which is probably due to two
main reasons: i) difficulties associated with generating 5 on a large
scale, which requires substantial amounts of the relatively unstable
protecting agent PMBOMCl, and ii) the requirement for low tem-
perature reactions and chromatographic purification during the syn-
KEY WORDS: histidine, N(), NAPOM, protecting group, race-
mization, peptide synthesis
10c
thesis of PMBOMCl. It should also be noted that the application
of the PMBOM group to the Boc-based strategy may be difficult
due to its lability under acidic conditions, which are required to re-
move the Boc group.
INTRODUCTION
We have recently reported that the 2-naphthylmethoxymethyl
(NAPOM) group (Chart 1) may serve as an alternative and also or-
Histidine (His; 1 in Scheme 1), one of the 20 naturally occurring
amino acids, contains a basic imidazole moiety with two nitrogen
atoms. The nitrogen atom that is in closer proximity to the stereo-
center will henceforth be denoted N() (from the Latin word “pros”
– “in front”), whereas the other will be referred to as N() (from the
Greek word “tele” – “far, far off”). In biologically active peptides
and proteins, the His imidazole moiety often serves as a hydrogen-
bonding and/or metal-binding site. Given these significant roles,
the controlled chemical synthesis of His-containing peptides has
become an important task in the synthetic community.
1
1
thogonally to PMB(OM). We discovered that the NAPOM group
is moderately stable in acidic media, i.e., it tolerates e.g. 1 equiva-
1
1
lent of camphorsulfonic acid in MeOH, and a catalytic amount of
1
2
trifluoromethanesulfonic acid at −20 °C, while it is cleaved with
1
1
4
CBr in refluxing MeOH. We envisaged that this lability of the
NAPOM group could potentially offer the chance of a selective re-
moval of the Boc group in the presence of NAPOM group. Further-
more, NAPOMCl, an introducing agent for the NAPOM group, is
a stable and storable solid that can be prepared at 0 °C without chro-
1
1
1
matographic purification. Herein, we report the chromatography-
and epimerization-free synthesis of N()-Fmoc- (6) or N()-Boc-
and N()-NAPOM-protected His (7), and their application to the
racemization-free synthesis of peptides.
Yet, the basicity of the imidazole moiety causes difficulties in the
2
peptide synthesis. Specifically, the His residue exhibits a high pro-
pensity toward epimerization, especially when its carboxylic moi-
ety is activated in e.g. acid-halides or acid-anhydrides (2; Scheme
1
), which is usually the case for the subsequent coupling with an
RESULTS AND DISCUSSION
elongating peptide chain. The epimerization is tentatively triggered
via the abstraction of a C-H by the -nitrogen to generate enolate
The first task of our project was the chromatography-free synthesis
of N()-NAPOM-protected 6 and 7 under retention of the high en-
3
, which affords L-His derivative 2 and its enantiomer ent-2 at the
3
same rate. In order to suppress this racemization, protection of the
more nucleophilic and hence more easily and selectively protecta-
ble -nitrogen atom with bulky and/or electron-withdrawing groups
antiomer excess (ee). The synthesis of N()-Boc derivative 7 com-
menced with the esterification of inexpensive 1 using SOCl and
2
MeOH to afford 8 (Scheme 2). Since this esterification is very clean,
crude 8, after complete evaporation of all volatiles, was used in the
next reaction, i.e., the selective introduction of the Boc group at the
4
such as triphenylmethyl (Trt) in e.g. 4 (Chart 1) was first attempted
5
to reduce the basicity of the -nitrogen atom indirectly. In fact, this
N()-protection considerably suppressed the racemization, albeit
-nitrogen atom of 8.
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Scheme 1. Main Epimerization Pathway of the Histidine
Residue During Chemical Peptide Synthesis.
Scheme 2. Chromatography-free synthesis of N()-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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5
5
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5
5
6
NAPOM-protected histidines 6 and 7.
0
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6
7
8
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9
0
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2
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5
6
7
8
9
0
Chart 1. Structures of protected histidines 4-7.
3
Initially, we very slowly added Boc O to a mixture of 8 and Et N
2
at room temperature (6 h), followed by stirring for further 18 h at
room temperature. This approach afforded 9a in excellent selectiv-
ity (9a : 9b : 10  97 : 3 : 0), but low ee, judging from the fact that
1
1 obtained from this lot exhibited only 33% ee. We envisaged that
Scheme 3. Racemization test: Liquid-phase synthesis of (A)
the racemization could potentially occur during the prolonged ex-
Boc-His(-NAPOM)-Pro-NH and (B) Fmoc-Ala-His-Pro.
2
posure of the N()-unprotected materials such as 8 and 9a to basic
3 2
conditions. Thus, 8 was subsequently exposed to Et N and Boc O
1
3
for only 2.5 h at room temperature, which furnished a mixture of
a, 9b, and 10 in a 89 : 9 : 2 ratio. The ensuing treatment of this
mixture with K CO in MeOH to cleave any unnecessary Boc
9
2
3
groups on the imidazole rings of 9a and 9b, generated Boc-His-
OMe 10 in 73% over three steps as the sole product. Subsequently,
1
2
0 was protected using Ac O to afford acetate 11 (91% yield) as a
colorless crystalline solid after reprecipitation. It should be noted
that attempts to purify 11 by recrystallization resulted in consider-
able racemization.
The introduction of the NAPOM group at the -nitrogen atom was
1
4
achieved by treating 11 with NAPOMCl in the presence of MS5A
at room temperature, and a subsequent saponification afforded
N()-Boc-, N()-NAPOM-protected His 7 in 50% yield over two
steps with 99.7% ee1 after recrystallization. Here, a special purifi-
cation process was required to obtain a pure specimen of 7: i) After
concentrating the organic solvent mixture from the saponification
in vacuo, the reagent-derived organic impurities were removed by
5
As chromatographic purification steps and halogenic solvents are
not required in this approach, our synthesis of N()-NAPOM-
protected His 6 and 7 could potentially be useful for the large-scale
(e.g. industrial) synthesis of such synthons. Indeed, the reproduci-
bility of this process was very good, at least on the gram scale. With
building blocks 6 and 7 in hand, we turned our attention to applying
them to the Boc- and Fmoc-based synthesis of peptides. To exam-
ine the racemization rate during Boc-based peptide synthesis
2
washing the aqueous layer with Et O under basic conditions, fol-
lowed by ii) a back extraction at pH  4, which recovered 7 in the
organic layer; iii) the organic layer was then evaporated to dryness;
iv) the residual crude material (84% purity, 90.2% ee) was dis-
solved in acetone and the undissolved solid, which is mainly a race-
mate of 7, was removed by filtration; v) the filtrate was evaporated
in vacuo, and the residue was recrystallized from hexane/acetone.
(
Scheme 3A), we selected Boc-His(-NAPOM)-Pro-NH
model peptide, considering that the His-Pro-NH sequence has pre-
viously been reported to racemize easily at the His residue during
2
(15) as a
Next, we moved on to the synthesis of Fmoc-protected His 6. The
remaining task to prepare 6 was a seemingly easy manipulation of
the protecting group (BocFmoc), which turned out to be more
complicated than anticipated. For instance, a known procedure us-
ing TMSOTf/2,6-lutidine for the removal of Boc was not suitable
2
6
,7
its synthesis. When N()-Z-N(-phenacyl-His (Z: benzyloxycar-
bonyl) or N()-Z-N(-phenacyl-His were coupled with Pro-NH
(16) using dicyclohexylcarbodiimide (DCC) in the presence of
Et N by Fletcher and co-workers, the target dipeptides were ob-
tained in 30% and 96% de, respectively. In contrast, our N-()-
NAPOM-protected His derivative 7 suppresses the racemization
significantly and affords the corresponding dipeptide 15 with
2
given that it requires the use of a halogenated solvent (CH
2
Cl
2
) and
3
6
gave impure 13 and thus 6 after introducing the Fmoc group, which
were difficult to purify; nevertheless, this procedure could be suc-
10ab
cessfully applied to the synthesis of the PMBOM counterpart 5.
15
After considerable experimentation, we ultimately found that the
use of HCl in ethyl acetate to give 14 and the use of N-(9-fluorenyl-
methoxycarbonyloxy)succinimide (FmocOSu) with Na CO fur-
2 3
nishes 6 (61% over 2 steps) without any byproducts detectable by
99.3% de.
For a demonstration of the synthetic utility in Fmoc-based liquid-
phase peptide synthesis (Scheme 3B), we chose Ala-His-Pro-OH
as a model peptide, as this sequence has previously been used in a
NMR analysis after workup. Indeed, this material showed, without
10ab
study to examine the racemization of PMBOM counterpart 5.
1
5
further purification, 97.7% purity and 99.7% ee via HPLC analy-
16
Fmoc-His()-NAPOM-OH 6 and Pro-O-t-Bu 17 were coupled us-
ing hexafluorophosphate azabenzotriazole tetramethyl uranium
sis.
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(
HATU) and diisopropylethylamine (DIPEA) to give Fmoc-pro-
(33.5 g) of diBOC compounds 9a and 9b, and a monoBoc com-
pound 10. This crude material was used directly in the next reaction
without further purification. In the air, K CO (1.26 g, 9.12 mmol)
2 3
1
2
3
4
5
6
7
8
9
tected dipeptide 18. After deprotection, free amine 19 was coupled
with Fmoc-Ala-OH 20 to yield protected tripeptide 21.
was added to the above crude mixture (33.5 g) in MeOH (360 mL)
at rt. After stirred under reflux for 1 h, the reaction mixture was
cooled to 0 °C, and quenched with saturated aqueous solution of
Finally, removal of both the NAPOM and t-Bu groups from 21 via
treatment with refluxing TFA in the presence of i-Pr
3
SiH and water
furnished tripeptide 22. Judging from the observed 99.7% de of
1
5
KHSO
mL) was added. The mixture was extracted with EtOAc (140 mL ×
9 times). The organic layer was washed with H O for 3 times, dried
over anhydrous Na SO , filtered and concentrated under reduced
4 2
(7.50 mL). The mixture was concentrated and H O (150
2
2, which was identical to the ee of amino-acid building block 6,
N()-NAPOM-protected His perfectly prevents the racemization,
even in the Fmoc-based strategy. It is noteworthy that when N()-
Trt-protected His counterpart 4 was subjected to the same peptide
synthesis conditions, the de of 22 decreased to 98.2% de (data not
shown).
2
2
4
pressure to give crude Boc-His-OMe 10 as a gum. To this gummy
crude material, a mixture of hexane and EtOAc (10:1) was added
and triturated to form powder material in the solvent. The powder
was collected by suction filteration to afford Boc-L-His-OMe 10
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
(19.08 g, 70.9 mmol, 73% for 3 steps) as a colorless powder. De-
Herein, we have reported a chromatography-free synthesis of N()-
NAPOM-protected histidine derivatives and demonstrated their ex-
tremely high ability to prevent racemization, both in the Boc- and
Fmoc-based synthesis of peptides. So far only very few N()-pro-
tecting groups are known to be compatible with both the Boc and
Fmoc strategies, despite continued demand of the Boc-based strat-
spite the absence of a purification process, the obtained powder
showed fully identical physical data to those reported in the litera-
1
8
25
ture, except for optical rotation. In our case, 10 showed [α]
D
D
2
5
+
13.1 (c 1.02, CHCl
9.70 (c 1.00, CHCl
3
), whereas the literature value was [α]
−
3
).18 We also measured the [α]
D
value of a com-
mercially available Boc-L-His-OMe (Watanabe Chemical Indus-
tries, LTD., Japan), whose ee had been confirmed as >99.9% ee by
1
7
egy in the base-sensitive synthesis of peptides. The BOM group
is one of these rare examples, albeit that it requires potentially ex-
plosive hydrogenolysis or corrosive HF, which may be problematic
in the context of a large-scale synthetic operation. Based on the re-
sults shown herein, it is feasible to expect a significantly increased
number of applications for the NAPOM protecting group.
2
6
HPLC (personal communication). The result was [α]
1.00, CHCl ), which is almost identical to ours.
Boc-L-His(τ-Ac)-OMe 11. In the air, Boc-L-His-OMe 10 (18.0
g, 66.8 mmol) was added to Ac O (90 mL) at 0 °C. After being
stirred at 0 °C for 1 h, the reaction mixture was concentrated, co-
evaporated with toluene (5 times), and triturated in Et O/hexane =
/5. The powder was collected by suction filtration to afford 11
18.9 g, 60.7 mmol, 91%) as a colorless powder. Despite the ab-
sence of a purification process, the obtained powder showed clean
D
+13.0 (c
3
2
2
1
(
EXPERIMENTAL SECTION
Amino acids, derivatives of amino acids, protecting and condensa-
tion agents were purchased from Watanabe Chemical Industries,
LTD, and used as supplied. Dehydrated toluene, MeOH, and N,N-
dimethylformamide (DMF) were purchased from Kanto Chemical
Co. Inc. or Wako Pure Chemical Industries Ltd., and used without
further dehydration. Molecular sieves 5A (MS5A) were preac-
tivated by heating in vacuo. All other chemicals were obtained from
local venders, and used as supplied. Thin-layer chromatography
1
29
1
H NMR. [α]
CDCl ) δ 8.04 (d, J = 1.6 Hz, 1H), 7.25 (s, 1H), 5.65 (broad d, J =
.6 Hz, 1H) 4.58–4.61 (m, 1H), 3.73 (s, 3H), 3.02–3.07 (m, 2H),
D
3
+28.8 (c 1.02, CHCl ); H NMR (400 MHz,
3
7
2
+
.56 (s, 3H), 1.43 (s, 9H); HRMS (ESI-TOF) m/z [M+Na] calcd
+
21 3 5
for C14H N NaO 334.1373, found 334.1359. Comment: An at-
tempt to recrystallize 11, via a procedure accompanied by heating,
resulted in the severe racemization. Because compound 11 seemed
unstable, it was soon used in the next reaction.
(
6
(
TLC) was performed using precoated TLC grass plates (silica gel
0 F254, 0.25 mm thickness) for the reaction analyses. Silica gel
spherical, neutral, 100–210 µm) was used for column chromatog-
Boc-L-His(π-NAPOM)-OH 7. After the moisture was removed
from Boc-L-His(τ-Ac)-OMe 11 (5.25 g, 16.9 mmol) by azeotropy
with dehydrated toluene (3 times), under argon atmosphere, dehy-
drated toluene (79.1 g) and MS5A (1.70 g, powder) were added and
the reaction mixture was cooled to 0 °C. To the mixture was added
NAPOMCl (5.23 g, 25.3 mmol) and the reaction was stirred at 0 °C
for 10 min, and at rt for 22 h. The reaction mixture was filtered and
the filtrate was evaporated under reduced pressure. The solid resi-
1
raphy. IR spectra were recorded on an FT/IR equipment. H and
13
C NMR spectra were recorded at 400 MHz. Chemical shifts are
reported in ppm from tetramethylsilane (TMS) with reference to
1
internal residual solvent [ H NMR, CDCl
3
(7.26), CD
3
OD (3.31),
DMSO-d6 (2.50); 1 C NMR, CDCl
3
(77.16), CD OD (49.00),
3
3
DMSO-d6 (39.52)]. The following abbreviations are used to desig-
nate the multiplicities: s = singlet, d = doublet, t = triplet, m = mul-
tiplet. High resolution mass spectra (HRMS) were recorded on an
ESI-TOF equipment.
2 2
due, which was poorly soluble in Et O, was washed with Et O (4
times) to give crude Boc-L-His(π-NAPOM)-OMe 12 (8.03 g) as a
colorless solid. The crude material was used directly in the next
reaction without further purification. In a stoppered flask, 2 M
aqueous solution of NaOH (21.1 mL) was added to a solution of
the above crude Boc-L-His(π-NAPOM)-OMe 12 (8.03 g) in MeOH
2
Boc-L-His-OMe 10. In the air, SOCl (9.00 mL, 125 mmol) was
added to a solution of L-histidine 1 (15.0 g, 96.7 mmol) in dehy-
drated MeOH (390 mL) at 0 °C. After being stirred at 80 °C for 16
h, the reaction mixture was concentrated, and the residue was co-
evaporated with MeOH for 3 times to give crude methyl ester 8
(
68 mL) at rt. After stirred at rt for 1.5 h, the reaction mixture was
diluted with H O (68 mL), and concentrated under reduced pres-
sure to remove MeOH. The residual aqueous mixture was washed
with Et O (70 mL × 3 times), and the pH of the aqueous layer was
adjusted to 4 using saturated aqueous solution of KHSO (6.2 mL).
The acidified aqueous mixture was extracted with AcOEt (70 mL
× 5 times). The combined organic layers were washed with H
(100 mL × 2 times) and dried over anhydrous Na SO4. The mixture
2
(23.42 g, colorless solid). The crude material was used directly in
the next reaction without further purification. In the air, to a solu-
tion of the above crude methyl ester 8 (23.42 g) in dehydrated
2
4
MeOH (390 mL), (Boc)
27.0 mL, 193 mmol) were added at 0 °C. After stirred at rt for 2.5
h, full consumption of methyl ester 8 was confirmed by TLC. The
reaction was quenched with saturated aqueous solution of NH Cl
150 mL). The mixture was concentrated, and the residue was ex-
tracted with EtOAc. The organic layer was washed with saturated
aqueous solution of NH Cl, dried over anhydrous Na SO , filtered
and concentrated under reduced pressure to give a crude mixture
2
O (46.4 g, 212 mmol) and triethylamine
(
2
O
2
4
was filtered and the filtrate was concentrated under reduced pres-
sure to give crude Boc-L-His(π-NAPOM)-OH 7 (6.76 g). To the
crude material was added acetone (338 mL) and the insoluble ma-
terial after sonication was filtered off. The filtrate was evaporated
and the resultant gummy residue was recrystallized from hexane /
acetone (2/3, 250 mL) to give Boc-L-His(π-NAPOM)-OH 7 (3.63
(
4
2
4
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g, 8.53 mmol, 50% for 2 steps) as colorless needles. The purity and
and the filtrate was evaporated in vacuo to give crude Boc-His(π-
NAPOM)-Pro-NH 15 (100.0 mg). After keeping a part of this
crude material (ca. 20 mg) for racemization analysis, the residue
was roughly purified by silica gel column chromatography, using
1
2
3
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9
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1
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4
5
5
5
5
5
5
5
5
5
5
6
the enantiomeric excess of 7 at this stage were determined by
HPLC as 97.7% (Figure S1) and 99.7% ee (Figure S2), respectively.
Comment: Although these purity and enantiomeric excess were
satisfactory to us, to ensure its commercialization, another recrys-
tallization using hexane / acetone (1/4, 150 mL) was examined. As
a result, purer specimen of 7 (2.80 g, 39%) was obtained as color-
less needles, whose purity and ee were 99.5% and 99.7% ee, re-
2
CHCl
3
and MeOH (50/1 to 10/1) as eluents, to give impure Boc-
15 to use as a reference of 15 in HPLC
His(π-NAPOM)-Pro-NH
2
analysis. In the same procedure, epi-15, whose His residue has D-
configuration, was synthesized from ent-7, and used in HPLC anal-
ysis (Figure S5A).
Fmoc-Ala-His-Pro-OH 22. In a stoppered flask, a solution of
Pro-OtBu 17 (94.0 mg, 0.548 mmol) in dehydrated DMF (1.65 mL)
was added to a solution of Fmoc-L-His(π-NAPOM)-OH 6 (200 mg,
0.365 mmol), HATU (278 mg, 0.731 mmol) and DIEA (63.6 µL,
0.365 mmol) in dehydrated DMF (2.0 mL) at rt. After stirred at rt
2
2
spectively (charts not shown). M.p. 148–149 °C; [α]
0.80, CHCl
); IR (neat) 3319 (broad), 3123, 2976, 2931, 1696 cm-
H NMR (400 MHz, CD OD) δ 8.18 (s, 1H), 7.87–7.77 (m, 4H),
.50–7.40 (m, 3H), 7.00 (s, 1H), 5.63 (d, J = 11.2 Hz, 1H), 5.59 (d,
J = 11.2 Hz, 1H), 4.75 (d, J = 12.0 Hz, 1H), 4.71 (d, J = 12.0 Hz,
D
+35.9 (c
3
1
1
;
3
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H), 4.39 (broad m, 1H), ca. 3.38–3.30 (m, 1H, overlapped with a
13
solvent peak); 3.08 (dd, J = 15.6, 8.4 Hz, 1H), 1.38 (s, 9H);
C
for 1 h, the reaction mixture was diluted with CHCl
solution was washed with saturated aqueous solution of KHSO
and then with that of Na CO . The organic layer was separated and
dried over anhydrous Na SO4. The mixture was filtered and the fil-
3
. The resultant
NMR (100 MHz, CD
3
OD) δ 175.4, 157.6, 138.4, 135.5, 134.7,
4
,
134.6, 131.1, 129.4, 129.0, 128.7, 127.9, 127.3, 127.2, 126.7, 125.3,
2
3
+
8
0.5, 76.1, 72.2, 54.8, 28.7, 27.7; HRMS (ESI-TOF) m/z [M+H]
2
+
calcd for C23
H
28
N
3
O
5
426.2023, found 426.2027. Through an
trate was concentrated under reduced pressure to give crude Fmoc-
His(π-NAPOM)-Pro-OtBu 18 (281 mg), which was used directly
in the next reaction without further purification. 20% piperidine so-
lution in dehydrated DMF (3.7 mL) was added to a solution of the
above crude Fmoc-His(π-NAPOM)-Pro-OtBu 18 (281 mg) in de-
hydrated DMF (0.3 mL) at rt. After stirred for 30 min, the reaction
mixture was concentrated under reduced pressure to give crude
His(π-NAPOM)-Pro-OtBu 19 (244 mg), which was used directly
in the next reaction without further purification. A solution of the
above crude His(π-NAPOM)-Pro-OtBu 19 (244 mg) in dehydrated
DMF (1.8 mL) was added to a solution of Fmoc-Ala-OH 20 (171
mg, 0.548 mmol), HATU (277.5 mg, 0.730 mmol) and DIEA (63.6
µL, 0.365 mmol) in dehydrated DMF (1.8 mL) at rt. After being
stirred at room temperature for 1.5 h, the reaction mixture was di-
identical method, ent-7 was synthesized from D-histidine. The
physical data of ent-7 were completely identical, except that it
showed negative optical rotation with keeping the absolute value.
Fmoc-L-His(π-NAPOM)-OH 6. In the air, 4 M HCl in EtOAc
(31.0 mL, 124 mmol) was added to a solution of Boc-L-His(π-
NAPOM)-OH 7 (2.00 g, 4.70 mmol) in EtOAc (16 mL) at 0 °C.
After stirred at rt for 1.5 h, the reaction mixture was concentrated
under reduced pressure. The residue was washed with EtOAc, and
concentrated to give crude L-His(π-NAPOM)-OH‧HCl 14 (2.03 g),
which was used directly in the next reaction without further purifi-
2 3
cation. In the air, saturated aqueous solution of Na CO (11.8 mL,
24.4 mmol) and FmocOSu (2.39 g, 7.05 mmol) were added to a
solution of the above-mentioned crude L-His(π-NAPOM)-OH‧HCl
1
4 (2.03 g) in THF (11.8 mL) at 0 °C. After stirred at rt for 16 h,
luted with CHCl
aqueous solution of KHSO
organic layer was separated and dried over anhydrous Na
mixture was filtered and the filtrate was concentrated under re-
duced pressure to give crude Fmoc-Ala-His(π-NAPOM)-Pro-OtBu
21 (305 mg), which was used in the next reaction without further
purification. To the above crude material 21 (69.5 mg) was added
a cocktail of i-Pr SiH (0.05 mL), H O (0.05 mL) and TFA (0.90
3 2
mL) at rt. After stirred under reflux for 1.5 h, the reaction was
cooled to rt and evaporated. The residual amorphus was washed
3
. The resultant solution was washed with saturated
, and then with that of Na CO . The
SO4. The
the reaction mixture was concentrated under reduced pressure. The
resulting aqueous mixture was washed with EtOAc (5 times), and
the pH was adjusted to 5.0 with saturated aqueous solution of
KHSO
times). The combined organic layers were washed with H
times) and dried over anhydrous Na SO4. The mixture was filtered
and the filtrate was concentrated under reduced pressure to give
crude Fmoc-L-His(π-NAPOM)-OH (2.65 g, colorless amorphus).
2
The crude material was triturated in Et O and the formed precipi-
4
2
3
2
4
. The acidified solution was extracted with EtOAc (10
2
O (4
2
tate was collected by suction filtration to furnish Fmoc-L-His(π-
NAPOM)-OH 6 (1.57 g, 2.87 mmol, 61%, 2 steps). The purity and
the ee were determined to be 97.7% and 99.7% ee, respectively, via
2
with Et O (3 times), to give crude Fmoc-Ala-His-Pro-OH 22 (48.0
mg). In the same procedure, crude material of epi-22, whose His
residue has D-configuration, was synthesized from ent-6, and used
as a reference in HPLC analysis (Figure S6A).
2
0
-
HPLC analyses (Figures S3 and S4). M.p. 171–172 °C; [α]
D
−
14.2 (c 0.30, DMF); IR (neat) 3407 (broad), 3132, 3056, 1710 cm
1
1
;
H NMR (400 MHz, DMSO-d6) δ 7.92–7.65 (m, 9H), 7.54–7.28
m, 7H), 6.80 (s, 1H), 5.51 (d, J = 11.0 Hz, 1H); 5.46 (d, J = 11.0
Hz, 1H), 4.59 (s, 2H), 4.39–4.16 (m, 3H), 3.45–3.27 (broad, 1H),
ASSOCIATED CONTENT
Supporting Information
HPLC charts to determine ee values, H and 13C NMR charts of all
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(
13
1
3
.24–3.11 (m, 1H), 3.01 (m, 1H); C NMR (100 MHz, DMSO-d6)
δ 173.0, 155.9, 143.8, 140.7, 138.5, 134.8, 132.8, 132.5, 127.9,
127.8, 127.7, 127.6, 127.1, 126.2, 126.0, 125.8, 125.3 × 2, 120.1,
7
+
3.4, 69.4, 65.7, 53.4, 46.6, 25.3; HRMS (ESI-TOF) m/z [M+H]
+
calcd for C33
H
30
N
3
O
5
548.2180, found 548.2196. Through an
AUTHOR INFORMATION
identical method, ent-6 was synthesized from D-histidine. The
physical data of ent-6 were completely identical, except that it
showed positive optical rotation with keeping the absolute value.
Corresponding Author
*E-mail: torikai@chem.kyushu-univ.jp
Boc-His(π-NAPOM)-Pro-NH
NH (13.9 mg, 0.117 mmol) was added to a solution of Boc-His (π-
NAPOM)-OH 7 (50.1 mg, 0.117 mmol), anhydrous HOBt (16.0 mg,
.117 mmol) and DCC (24.4 mg, 0.117 mmol) in dehydrated DMF
1.8 mL) at 0 °C. After stirred at rt for 16 h, the reaction mixture
was filtered and the filtrate was concentrated under reduced pres-
sure. After added water, the mixture was extracted with CHCl . The
organic portions were combined and washed with saturated aque-
ous Na CO . The organic layer was dried over Na SO and filtered,
2
15. In a stoppered flask, Pro-
ORCID
2
Kohei Torikai: 0000-0002-9928-4300
0
(
Notes
The authors declare that a patent regarding the use of NAPOMCl
has been granted in the USA (Kyushu University, Agent for Intro-
ducing Protecting Group for Hydroxy group and/or Mercapto
3
2
3
2
4
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Page 5 of 5
Organic Process Research & Development
Group, Patent No. US10160694, December 25, 2018), and is pend-
Fmoc-His(Mmt)-OH und Fmoc-His(Mtt)-OH. Zwei Nue Histidin-
im
derivative N -Geschützt mit Säure-hochempfindlichen Gruppen.
1
2
3
4
5
6
7
8
9
ing in several other countries (Kyushu University, Agent for Intro-
ducing Protecting Group for Hydroxy group and/or Mercapto
Group, Publication No. 20170305809, October 26, 2017).
Darstellung, Eigenschaften und Einsatz in der Peptidsynthese, Tetrahedron
Lett. 1991, 32, 475–478.
(6) Fletcher, A. R.; Jones, J. H.; Ramage, W. I.; Stachulski, A. V. The Use
of the N(π)-Phenacyl Group for the Protection of the Histidine Side Chain
in Peptide Synthesis, J. Chem. Soc., Perkin Trans. 1 1979, 2261–2267.
ACKNOWLEDGMENTS
(7) Kimbonguila, A. M.; Boucida, S.; Guibé, F.; Loffet, A. On the Allyl
This work was financially supported by JSPS KAKENHI grant
JP15K21210 for Young Scientists (B) (to K.T.), and a grant from
Wako Pure Chemical Industries (to K.T.) related to a Wako Award
in Synthetic Organic Chemistry (Japan).
Protection of the Imidazole Ring of Histidine, Tetrahedron 1997, 53,
12525–12538.
(8) Brown, T.; Jones, J. H.; Richards, J. D. Further Studies on the Protection
of Histidine Side Chains in Peptide Synthesis: The Use of the π-
Benzyloxymethyl Group, J. Chem. Soc., Perkin Trans. 1 1982, 1553–1561.
(9) Colombo, R.; Colombo, F.; Jones, J. H. Acid-labile Histidine Side-
chain Protection: The N(π)-t-Butoxymethyl Group, J. Chem. Soc. Chem.
Commun. 1984, 292–293.
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and Peptidomimetics (Houben-Weyl E22a: Methods of Organic
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(
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(
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3
2
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8
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im
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