Here, the synthesis and applications of (E)-ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate as a racemization suppressing and easily recyclable version of the Yamaguchi reagent that can be used for amide and peptide synthesis are reported. We demonstrated its application in racemization-free esterification, thioesterification, amidation, and peptide bond formation. We successfully synthesized oligopeptides on the solid support in dimethylformamide as well as in solution (dichloromethane) by applying this coupling reagent. It is important to note that a mixed-anhydride-based method provides peptide-forming reactions as good as the current methods using built-in coupling reagents. Mechanism investigation, racemization suppression, and recyclability are also discussed.
Here, the synthesis and applications of (E)-ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate as a racemization suppressing and easily recyclable version of the Yamaguchi reagent that can be used for amide and peptide synthesis are reported. We demonstrated its application in racemization-free esterification, thioesterification, amidation, and peptide bond formation. We successfully synthesized oligopeptides on the solid support in dimethylformamide as well as in solution (dichloromethane) by applying this coupling reagent. It is important to note that a mixed-anhydride-based method provides peptide-forming reactions as good as the current methods using built-in coupling reagents. Mechanism investigation, racemization suppression, and recyclability are also discussed.
Esterification
and amidation are extensively used in the synthesis
of natural products, polymers, active pharmaceutical ingredients,
and other biologically relevant molecules.[1,2] These
reactions are usually achieved by activation of a carboxylic acid
by a suitable coupling reagent. To date, many efficient coupling reagents
have been developed, including carbodiimides, phosphonium, and uronium/aminium
salts.[3] Some other carboxylic acid activation
methodologies have also been established, for example, acyl chlorides
in the Schotten–Baumann reaction,[4] azides in the Staudinger reaction,[5] activated
esters,[6] metal-catalyzed condensation reactions,[7] Lewis acid catalysis,[8] etc.Similarly, mixed-anhydride-mediated coupling reactions
are also
widespread.[9] In this direction, Yamaguchi
and co-workers reported a coupling reagent, 2,4,6-trichlorobenzoyl
chloride (TCBCl or Yamaguchi reagent),[10] for efficient synthesis of esters and lactones via in situ formation
of mixed anhydrides in 1979. Later, Shiina and co-workers introduced
the use of aromatic anhydrides, especially, 2-methyl-6-nitrobenzoicanhydride[11] as a better alternative to
the Yamaguchi reagent. A catalytic amount of 4-dimethylaminopyridine
(DMAP) was used with the Yamaguchi reagent as well as with the aromatic
anhydrides to improve the regioselectivity.[12] In 2014, a modified Yamaguchi reagent was reported by Yoshinori
et al. They introduced 2,4,6-trichlorobenzoyl-4-dimethylaminopyridinium
chloride (TCB-DMAP)[13] as the coupling reagent
to avoid the use of often intractable acid chlorides and acid anhydrides,
which hinders their use in the presence of sensitive functional and
protecting groups. These reagents are capable of affording condensation
reactions efficiently, but they cause a significant amount of racemization
in case of chiral substrates. Thus, the yield of the desired products
decreases and purification of the products becomes cumbersome. Most
importantly, these reagents become useless for the stepwise synthesis
of oligopeptides because of the accumulation of undesired epimerized
analogues at each step. Usually, N-hydroxy amine
reagents, for example, hydroxybenzotriazole (HOBt) and hydroxyazabenzotriazole
(HOAt), are frequently used as additive with the coupling reagents[14] to prevent the racemization during peptide synthesis.
However, due to the explosive nature of HOBt and HOAt, recently, ethyl-2-hydroxyimino-2-cyanoacetate
(oxyma) has been suggested as a racemization suppressant in diisopropylcarbodiimide[15] and oxyma-based phosphate[16]-mediated peptide syntheses. Earlier, we also reported the
utility of oxyma-based reagents for racemization-free peptide synthesis
and various other organic transformations.[17]We describe herein a differently modified Yamaguchi reagent,
(E)-ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate
(Scheme , TCBOXY, I), which can be efficiently used for esterification, amidation,
and peptide synthesis without causing detectable racemization. To
the best of our knowledge, to date, there is no report on the racemization-free
amide and peptide synthesis using Yamaguchi and modified Yamaguchi
reagents.
Scheme 1
Preparation of the Coupling Reagent, Ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate
(TCBOXY, I)
Results and Discussion
Reagent I can readily
be synthesized by reacting oxyma
with TCBCl in the presence of Hunig’s base (N,N-diisopropylethylamine (DIPEA)) under nitrogen
atmosphere and dry dichloromethane (DCM) at 0 °C for 2 h (Scheme ). Simple aqueous
workup and recrystallization from hexane results in I that can be directly used for coupling reactions. Reagent I does not degrade at room temperature (25 °C) and therefore
it can be stored for an extended period. A time-dependent high-performance
liquid chromatography (HPLC) study indicated no change of I until 2 months (Figures S4 and S5, Supporting
Information).Its coupling efficiency was first investigated
by the reaction
between benzoic acid and cyclohexylamine in the presence of DIPEA
(Table ). We observed
only 30% of the desired product and 60% of the corresponding amide
of 2,4,6-trichlorobenzoic acid (Figures S154–S156, Supporting Information). To avoid the formation of side products,
we used a catalytic amount of DMAP, which acted as a selective acyl-transfer
reagent.[18] We screened the various amounts
of DMAP using the same reaction in DCM (Table ). We observed a 91% yield of the desired
product with 0.3 equiv of DMAP. By increasing the amount of DMAP beyond
0.3 equiv, no notable improvement in the yield was observed (entries 1 and 2). But, when we decreased the same below
0.3 equiv, the yield dropped (entries 4 and 5). Next, we screened several solvents using reagent I (1 equiv) with the catalytic amount of DMAP (0.3 equiv). We found
that DCM, CH3CN, EtOAc, CHCl3, and dimethylformamide
(DMF) afforded very good yields (entries 3 and 7–10). However, there was no reaction
in CH3OH and H2O (entries 13 and 14). Therefore, 0.3 equiv of DMAP and DCM as a solvent were
accepted under optimized conditions (entry 3).
Table 1
Optimization of Reaction Conditionsa
entry
solvents
DMAP (equiv)
yieldb (%)
1
DCM
0.8
91
2
DCM
0.4
91
3
DCM
0.3
91
4
DCM
0.2
80
5
DCM
0.1
65
6
DCM
0.0
30
7
CH3CN
0.3
80
8
EtOAc
0.3
72
9
CHCl3
0.3
75
10
DMF
0.3
85
11
THF
0.3
59
12
toulene
0.3
40
13
MeOH
0.3
0
14
H2O
0.3
0
Reaction conditions:
benzoic acid
(122 mg, 1 mmol), TCBOXY (349 mg, 1 mmol), DIPEA (193 mg, 1.5 mmol),
DMAP, cyclohexylamine (99 mg, 1 mmol) stirred at room temperature
for 15 min.
Isolated yield.
Reaction conditions:
benzoic acid
(122 mg, 1 mmol), TCBOXY (349 mg, 1 mmol), DIPEA (193 mg, 1.5 mmol),
DMAP, cyclohexylamine (99 mg, 1 mmol) stirred at room temperature
for 15 min.Isolated yield.Under the optimized conditions,
we proceeded to investigate the
scope of esterification using various carboxylic acids and alcohols
by using I. Reactions worked well with the sterically
hindered amino acids (Scheme , 2a–d) as well as with heterocyclic,
aromatic, and aliphatic carboxylic acids (2e–j). The reaction also worked with secondary alcohol (2h) in very good yield. We also extended this protocol for
thioesterification (2k–n). A broad
range of carboxylic acids was tolerated, including those bearing electron-donating
groups (2l and 2n) and an electron-withdrawing
group (2m), with aromatic thiols bearing neutral, electron-donating,
and electron-withdrawing substituents.
Scheme 2
Esterification, Thioesterification,
and Amidation by Using I
Reaction
conditions: acid (1
mmol), I (1 mmol), DIPEA (1.5 mmol), DMAP (0.3 mmol),
and alcohol, thiol, or amine (1.2 mmol), stirred at room temperature
for 5–30 min. Isolated yields after column chromatographic
purification are mentioned.
Esterification, Thioesterification,
and Amidation by Using I
Reaction
conditions: acid (1
mmol), I (1 mmol), DIPEA (1.5 mmol), DMAP (0.3 mmol),
and alcohol, thiol, or amine (1.2 mmol), stirred at room temperature
for 5–30 min. Isolated yields after column chromatographic
purification are mentioned.Further, we extended
this protocol for amidation reactions. Interestingly,
the reactions worked well with the aromatic carboxylic acids (Scheme , 2o–x), aliphatic carboxylic acids (2y–z), aromatic amines (2o), aliphaticamines (2p–w and 2z),
and C-protected amino acids (2x and 2y).We further explored the applicability of I for peptide
synthesis in solution with various N-protected amino acids bearing
various side chains (Scheme ). The reactions worked well with the common N-protections,
such as Bz (Scheme , 3a), Fmoc (3b to rac-3n),
Boc (3o and 3p), Cbz (3q–s), and sterically hindered amino acids with good to excellent
yields.
Scheme 3
Wide Scope of the Synthesis of Peptides in Solution Using I
Reaction conditions: acid (1
mmol), I (1 mmol), DIPEA (1.5 mmol), DMAP (0.3 mmol),
and amine (1.5 mmol), stirred at room temperature for 20–120
min. Isolated yields after column chromatographic purification are
mentioned.
Wide Scope of the Synthesis of Peptides in Solution Using I
Reaction conditions: acid (1
mmol), I (1 mmol), DIPEA (1.5 mmol), DMAP (0.3 mmol),
and amine (1.5 mmol), stirred at room temperature for 20–120
min. Isolated yields after column chromatographic purification are
mentioned.We further synthesized a tetrapeptide,
Boc-Val-Val-Ile-Ala-OMe,
the C-terminal segment of the amyloid β-peptide[20] (Figure a), in DCM solution following Boc-chemistry. The yield of the crude
peptide (after precipitation with cold ether but before purification
by HPLC) was 75%, while the purity was 99%, as determined by reversed-phase
(RP)-HPLC analysis (Figures S195 and S196, Supporting Information), indicating cleanliness of the procedure.
Later, we synthesized acyl carrier protein (ACP) (24–33) peptide
segment (ACP, acyl carrier protein: Asp-Asn-Ala-Ser-Phe-Val-Glu-Asp-Leu-Gly-NH2, Figure b)
and the tropoelastin peptide segment ((Pro-Gly-Val-Gly-Val-)2-NH2, Figure c)[21] by stepwise coupling of amino
acids on the Rink Amide MBHA resin following Fmoc/t-Bu orthogonal protection strategy. The electrospray ionization (ESI)-mass
spectrometry (MS) data and HPLC profiles of each segment during the
synthesis of ACP (24–33) (Table S1 and Figures S201–S218, Supporting Information) indicate
occurrences of neither incomplete coupling nor side reactions during
the coupling steps. Considering the scale of the reaction sequences,
yields (21% for ACP (24–33) and 30% for the elastin peptide)
with respect to the resin loading were good (Figures S217, S218 and S199, S200). We further synthesized specific segments of gramicidin A, B, and C on Wang resin (Figure d). The general structure of the segments of gramicidin A, B, and C is Xaa-d-Leu-l-Trp-d-Leu-l-Trp-OH, where the side chain
of Xaa varied (Figure d). We obtained these peptides also in good yield, i.e., 23, 28,
and 26% of gramicidin A, B, and C segments, respectively, with respect to the resin loading after
purification using RP-HPLC (Figures S219–S224, Supporting Information).
Figure 1
Sequences of the synthesized long peptides:
(a) Boc-VVIA-OMe, in
solution; (b) DNASFVEDLG-NH2; (c) PGVGVPGVGV-NH2; and (d) segments of gramicidin A, B,
and C using solid phase
peptide synthesis strategy.
Sequences of the synthesized long peptides:
(a) Boc-VVIA-OMe, in
solution; (b) DNASFVEDLG-NH2; (c) PGVGVPGVGV-NH2; and (d) segments of gramicidin A, B,
and C using solid phase
peptide synthesis strategy.Epimerization during such syntheses is highly important for
industry
and academia, but we could not find any systematic study on the epimerization
potential of such reagents. Therefore, we investigated the epimerization
potential of the Yamaguchi and related reagents and compared it to
that of I. We first synthesized dl-Fmoc-Phe-OBn
(rac-2a) using the optimized protocol and passed through
a chiral column. Two well-separated peaks, corresponding to the two
enantiomers, were observed in HPLC profile (Figures and S157–S159, Supporting Information) that were used as a reference. Next, we
synthesized l-Fmoc-Phe-OBn (2a) using the Yamaguchi
reagent in the absence of DMAP and in the presence of DMAP, the modified
Yamaguchi reagent (TCB-DMAP), and I. The products were
passed through the chiral column using the same eluant. Although all
of the tested reagent combinations resulted in 12–24% epimerization, I did not cause any detectable epimerization (Figures and S160–S166, Supporting Information). This result indicates that no racemization
occurred during the coupling reaction using I, unlike
other similar reagents. The above results also suggest that DMAP has
no apparent role in racemization suppression.
Figure 2
HPLC images to compare
the epimerization caused by various coupling
reagents.
HPLC images to compare
the epimerization caused by various coupling
reagents.We synthesized Fmoc-l-Ala-l-Leu-OMe (3g) and Fmoc-dl-Ala-l-Leu-OMe (3h) using I to investigate
the associated racemization in peptide synthesis.
The appearance of the single peak in the HPLC profile of 3g corresponds to the unique stereoisomeric product, whereas the presence
of the twin peak in the same of 3h indicates the presence
of two diastereomeric products (Figures and S167–S171, Supporting Information). The 1H and 13C NMR
spectra of 3h and 3g were also compared
(Figures and S106–S111, Supporting Information). We
found one singlet at δ = 3.72 ppm for the methoxy proton of 3g and two singlets at δ = 3.70 and 3.68 ppm for the
methoxy proton of 3h in the 1H NMR spectra.
Similarly, in the 13C NMR spectra, we found two peaks at
δ = 173.4 and 172.3 ppm corresponding to the two carbonyls of
the amide and the ester groups of 3g, indicating the
presence of the single diastereomer, whereas the presence of four
peaks at δ = 173.4, 173.3, 172.5, and 172.4 ppm for those carbonyl
carbons of 3h indicates the presence of two diastereomeric
products (Figure ).
Therefore, it was inferred that no detectable racemization occurred
during the synthesis of the mentioned dipeptides using I. Similarly, comparison of the HPLC profiles (Figures S172–S176, Supporting Information) of the l and dl forms of the Fmoc-Phe-Gly-OMedipeptides (3n and rac-3n, respectively) leads to the same
conclusion. Also, for all of the remaining l,l-dipeptides
depicted in Scheme , single peaks corresponding to the only stereoisomeric products
were noted, confirming the occurrence of no detectable racemization
during peptide synthesis by I.
Figure 3
Comparative study of
racemization by HPLC, 1H NMR, and 13C NMR of
Fmoc-l-Ala-l-Leu-OMe (left panel)
and Fmoc-dl-Ala-l-Leu-OMe (right panel).
Comparative study of
racemization by HPLC, 1H NMR, and 13C NMR of
Fmoc-l-Ala-l-Leu-OMe (left panel)
and Fmoc-dl-Ala-l-Leu-OMe (right panel).Next, we synthesized a tripeptide Z-Gly-Phe-Val-OMe
(Scheme , 3s and Figures S145–S147, S177 and S178, Supporting
Information) in solution using I, determined the yield
and the degree of racemization, and compared the results to those
reported for popular coupling reagents, such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-ethylmethanaminium hexafluorophosphate N-oxide (HATU), 1-((dimethylamino)-(morpholino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridinium
hexafluorophosphate-3-oxide (HDMA), 1-((dimethylamino)(morpholino)methylene)-1H-benzotriazolium
hexafluorophosphate-3-oxide (HDMB), and N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene] N-methylmethanaminium hexafluorophosphate N-oxide (HBTU).[19] Although the yields were
comparable, no racemization could be observed during the synthesis
by using I unlike the other reagents (Table ).
Table 2
Comparison
of the Yield and Racemization
of Z-Gly-Phe-Val-OMe Synthesized Using Various Coupling Reagents
entry
coupling reagent
yield (%)
racemization
(%)
1
HDMB
90
2.9
2
HDMA
90
0.7
3
HBTU
89
5.9
4
HATU
90
1.6
5
TCBOXY
90
n.d.a
No racemization
could be detected.
No racemization
could be detected.Next,
we turned our attention to the mechanism elucidation. As
suggested in the original article, Yamaguchi reagent, which is a sterically
hindered benzoyl chloride, works via the formation of the mixed-anhydride
intermediate[10] (II, Scheme ), followed by the
nucleophilic attack of DMAP to the less hindered electrophilic center
of II to generate the resonance-stabilized N-acyl pyridinium salt intermediate III.[13] Such preference of DMAP results in the regioselectivity
of the reaction. Further attack of the added nucleophile, e.g., alcohol
or amine, leads to the final product. However, SantaLucia and Dhimitruka[12] suggested that aliphatic carboxylates should
be more reactive toward the aliphatic moiety of the mixed anhydrideII due to the steric factor. Thus, once II is
formed, the less hindered carboxylate reacts with it to generate the
relatively less hindered symmetrical anhydride intermediate (IV). They indeed demonstrated that the byproduct, 2,4,6-trichlorobenzoate
anion, did not react with the Yamaguchi reagent to form 2,4,6-trichlorobenzoicanhydride, instead intermediate IV was generated exclusively.
Scheme 4
Plausible Mechanism of the Reaction Mediated by I
Therefore, most probably in
the TCBOXY-mediated reaction, the nucleophile
generated by the deprotonation of the substrate carboxylic acid in
the presence of DIPEA attacks the carbonyl carbon of I, forming the mixed anhydrideII, and releases the resonance-stabilized
oxyma anion. Then, another molecule of the already created carboxylate
attacks the less hindered carbonyl of II to generate IV. DMAP attacks regioselectively at the less hindered carbon
atom of either II or IV, forming the N-acyl pyridinium saltIII. The released oxyma
anion then attacks the carbonyl carbon of III, resulting
in the formation of the intermediate V, which is the
oxyma ester of the substrate carboxylic acid. V then
undergoes nucleophilic substitution in a stereoselective fashion to
produce the corresponding esters, thioesters, amides, and peptides.
The intermediate V was isolated as a product in a similar
reaction of a carboxylic acid (Fmoc-Ala-OH), I, the appropriate
amount of DMAP and DIPEA, but devoid of the nucleophile. It was characterized
by 1H NMR and 13C NMR spectroscopies (Figures S6 and S7, Supporting Information).Chemical waste generation and nonrecyclability are common problems
for the majority of the popular peptide-coupling reagents. Therefore,
a recent trend is to develop easily recyclable coupling reagents that
are highly required for sustainability.[22] We investigated the recyclability of I. We repeated
the synthesis of 2v (Scheme ) for that. After completion of the reaction,
the product and byproducts, oxyma (a) and trichlorobenzoic
acid (b) (Scheme and Figures S148–S153,
Supporting Information), were purified by eluting with specific eluents
from a silica gel column. The recovered b was chlorinated
with thionyl chloride by heating at 110–114 °C in toluene
for 3 h and mixed with the recovered a in the presence
of DIPEA to obtain I with 52% overall yield with respect
to the initial I used. Alternatively, the recovered a and b were recombined by merely heating in
the presence of silica gel under microwave irradiation[23] (Scheme )-based dehydration without using unhealthy thionyl chloride.
By this way, we were able to recover byproducts and recombine to regenerate
the coupling reagent easily.
Scheme 5
Recyclability of the Coupling Reagent,
TCBOXY
Conclusions
Yamaguchi
reagent could solve a lot of practical problems, but
to the best of our knowledge, could never be used as a peptide-coupling
reagent, primarily because of the arduous acid chloride and significant
racemization during condensation. We have developed a modified Yamaguchi
reagent, TCBOXY (I), which can be efficiently used for
the syntheses of esters, thioesters, amides, peptides in both solution
and solid support with very good to excellent yield. The advantages
of TCBOXY are as follows: (a) its preparation protocol is easy and
involves single-step reaction; (b) it suppresses racemization during
the coupling reaction and allows enantioselective syntheses; (c) only
2,4,6-trichlorobenzoic acid and oxyma are generated as byproducts,
which are nontoxic and can be recovered and recycled to generate the
same coupling reagent easily that can be used in the same pool; (d)
operationally simple, as both of the byproducts are acidic in nature
and just basic workup renders pure products. Thus, the described method
for the synthesis of the mentioned compounds using this new reagent
is a more eco-friendly and green process than the current alternatives
discussed in Introduction.
Experimental
Section
General Information
All of the reagents, except those
mentioned, were procured from usual commercial sources. NMR experiments
were performed on 600 and 400 MHz spectrometers using CDCl3. Tetramethylsilane was used as an internal standard. Chemical shifts
(δ) were indicated in parts per million. Spin–spin coupling
constants (J) were indicated in hertz. The multiplicity
of the signals was indicated as follows: s (singlet), d (doublet),
t (triplet), q (quartet), and m (multiplet). Thin-layer chromatography
using silica gel G254 was used to monitor the reactions. Column chromatography
using silica gel (60–120 mesh) and EtOAc/hexane as eluent was
used to purify the products. Melting points and Fourier transform
infrared (FT-IR) spectra were recorded on a dedicated meting point
apparatus and an FT-IR spectrometer, respectively. High-resolution
mass spectrometry (HRMS) experiments were performed on a Micromass
quad time-of-flight (Q-TOF) ESI-MS instrument and a Q-TOF liquid chromatography/MS
system. HPLC analyses were performed with reversed-phase chromatographic
columns and CHIRAL PAK AS-H (5 μm, 4.6 × 250 mm2) column attached to an UV detector. HPLC analyses were performed
with HPLC-grade solvents.
Procedure for the Synthesis of the Coupling
Reagent (E)-Ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate
(TCBOXY, I)
DIPEA (129 mg, 1 equiv) was added
to a solution of oxyma (142 mg, 1 equiv) in 2 mL of DCM under nitrogen.
The temperature of the reaction mixture was decreased to 0 °C.
Then, 2,4,6-trichlorobenzoyl chloride (243 mg, 1 equiv) was added
dropwise. The mixture was then stirred at room temperature for another
2 h. After completion of the reaction, 10 mL of DCM was added to it
and washed with 5% HCl (3 × 5 mL). Finally, the organic portion
was collected, dried using anhydrous CaCl2, and evaporated.
The obtained solid mass was recrystallized with hexane. Rf: 0.50 (EtOAc/hexane, 1:9); yield 326 mg, 94%; white
crystalline solid, mp 83–85 °C; 1H NMR (600
MHz, CDCl3): δ 7.47 (s, 2H), 4.54–4.53 (q, J = 7.2 Hz, 2H, CH2), 1.47–1.44 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 158.7, 156.6, 138.6, 134.0, 132.8, 128.7, 127.9, 106.6,
65.0, 14.1; IR (KBr) 3076, 1806, 1732, 1577, 1375, 1220, 1150, 1087,
990 cm–1; HRMS (ESI) m/z: [M + Na]+ calcd for C12H7Cl3N2NaO4 370.9369, found 370.9367.
Procedure for the Synthesis of Esters, Thioesters, and Amides
TCBOXY (1 equiv) was added to a DCM (2 mL) solution of carboxylic
acid (1 equiv), DMAP (0.3 equiv), and DIPEA (1.5 equiv). All of the
components were mixed well for 3–5 min for preactivation, and
then, alcohol, thiol, or amines (1.2 equiv) was added to it. Then,
the mixture was stirred for 5–30 min at room temperature. After
completion of the reaction, the whole mixture was diluted with 20
mL of ethyl acetate. The organic portions were collected, washed with
5% HCl (3 × 5 mL) and 5% NaHCO3 (3 × 5 mL), and
dried using anhydrous Na2SO4. Finally, Na2SO4 was filtered off and the solvent was evaporated
to obtain the product, which was purified by column chromatography.
Procedure for the Peptide Synthesis
TCBOXY (1 equiv)
was added to a solution of N-protected amino acid (1 equiv), DMAP
(0.3 equiv), and DIPEA (1.5 equiv) in 2 mL of DCM. The reaction mixture
was stirred for 5 min for preactivation, followed by the addition
of methyl ester of amino acid (1.5 equiv) and DIPEA (1.5 equiv) in
1 mL of DCM. The reaction mixture was stirred at room temperature
for 20–120 min. After completion of the reaction, the reaction
mixture was diluted with 20 mL of ethyl acetate; the organic phase
was washed with 5% HCl (3 × 5 mL), 5% NaHCO3 (3 ×
5 mL), and brine; and dried using anhydrous Na2SO4. Finally, Na2SO4 was filtered and the solvent
was evaporated. The product was purified by silica gel column chromatography.
Solution-Phase Synthesis of Boc-VVIA-OMe
TCBOXY (1
equiv) was added to a solution of Boc-Ile-OH (1 equiv), DMAP (0.3
equiv), and DIPEA (1.5 equiv) in 2 mL of DCM. The reaction mixture
was stirred for 5 min for preactivation. In another RB, methyl ester
of alanine (1.5 equiv) was taken in DCM and DIPEA was added to it
until basic pH was reached. Finally, this solution was added to the
above solution and stirring was continued until completion of the
reaction. Then, the reaction mixture was diluted by 20 mL of EtOAc
and washed by 5% NaHCO3 solution (2 × 5 mL) and 5%
citric acid solution (2 × 5 mL). Finally, the combined organic
layer was dried using anhydrous Na2SO4. The
solid product (Boc-IA-OMe) was obtained after evaporation of EtOAc
by a rotary vacuum evaporator.In 50 mL of RB, solid product
(Boc-IA-OMe) was taken and TFA/DCM (1:1) mixture was added and stirred
up to 2.5 h. After that, TFA was evaporated by a rotary vacuum evaporator,
the solution was washed three to four times with diethyl ether, and
finally a white solid (IA-OMe) was obtained. After Boc deprotection,
the resulting IA-OMe was coupled with Boc-V-OH following the procedure
as mentioned earlier to obtain Boc-VIA-OMe. Another cycle of Boc-deprotection and coupling with Boc-V-OH resulted
in white solid Boc-VVIA-OMe, which was characterized by reversed-phase
HPLC, with a retention time 4 min on a linear gradient of 0–70%
for 0–10 min and then 70–100% for 10–25 min CH3CN in H2O with 0.1% formic acid in a symmetry C8
analytical column. Low-resolution mass spectrometry (LRMS) (ESI) m/z: [M + H]+ calcd for C25H47N4O7 515.3445, found
515.3521. The yield was 75% with respect to starting material Boc-isoleucine.
Solid-Phase Synthesis of PGVGVPGVGV-NH2 and DNASFVEDLG-NH2
Decapeptide was manually assembled stepwise on Fmoc
Rink Amide MBHA resin by Fmoc/tert-butyl (t-Bu) protection strategy. Fmoc amino acids (1.5 equiv),
TCBOXY (1 equiv), DMAP (0.3 equiv), and DIPEA (3 equiv) were kept
for preactivation for 5 min. Then, amino acid coupling was performed
for 2–4 h. Fmoc deprotection was carried out using TFA/DCM
(1:1) mixture for 2.5 h. Purification of the peptide was carried out
by preparative HPLC, and lyophilization afforded the final peptide.
Solid-Phase Synthesis of Segments of Gramicidin A, B, and C
The syntheses were
carried out by stepwise coupling of amino acids on Wang resin, as
mentioned before, by Fmoc/t-Bu protection strategy.
Fmoc amino acids (1.5 equiv), TCBOXY (1 equiv), DMAP (0.3 equiv),
and DIPEA (3 equiv) were kept for preactivation for 5 min. Then, amino
acid coupling was performed for 2–4 h. Fmoc deprotection was
carried out using TFA/DCM (1:1) mixture for 2.5 h. Purification of
the peptide was carried out by preparative HPLC, and lyophilization
afforded the final peptide.
Procedure to Identify (E)-ethyl-9-cyano-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,7-dioxa-4,8-diazadec-8-en-10-oate
(Intermediate V, Scheme )
TCBOXY (1 equiv) was added to a solution of Fmoc-Ala-OH
(1 equiv), DMAP (0.3 equiv), and DIPEA (1.5 equiv) in 2 mL of DCM.
The reaction mixture was stirred for 30 min at room temperature. After
30 min, we observed one spot in TLC. The reaction mixture was diluted
with 20 mL of ethyl acetate; the organic phase was washed with 5%
HCl (3 × 5 mL), 5% NaHCO3 (3 × 5 mL), and brine;
and dried using anhydrous Na2SO4. Finally, Na2SO4 was filtered off and the solvent was evaporated
to obtain the intermediate, which was purified by column chromatography. Rf: 0.50 (EtOAc/hexane, 2:8); yield 357 mg, 82%;
white solid, mp 95–97 °C; 1H NMR (600 MHz,
CDCl3): δ 7.77–7.76 (d, J = 7.8 Hz, 2H), 7.61–7.59 (t, J = 6.6 Hz,
2H), 7.41–7.39 (t, J = 7.2 Hz, 2H), 7.33–7.31
(t, J = 7.2 Hz, 2H), 5.38 (br s, 1H), 4.43–4.36
(m, 3H), 4.24–4.20 (m, 3H), 1.44–1.43 (d, J = 7.2 Hz, 3H), 1.29–1.28 (m, 3H); 13C NMR (150
MHz, CDCl3) δ 173.3, 155.8, 144.0, 141.5, 127.9,
127.3, 125.3, 120.2, 67.2, 61.8, 49.9, 47.4, 19.0, 14.3; IR (KBr)
2925, 1747, 1692, 1534, 1450, 1260, 1027, 738 cm–1.
Recyclability of the Coupling Reagent, TCBOXY
TCBOXY
(1 equiv) was added to a solution of 2-picolinic acid (1 equiv), DMAP
(0.3 equiv) and DIPEA (1.5 equiv) in 2 mL of DCM. The reaction mixture
was stirred for 3–5 min for preactivation, followed by the
addition of tert-butyl amine (1.2 equiv). The reaction
mixture was stirred at room temperature for 45 min. After completion
of the reaction, the reaction mixture was diluted with ethyl acetate
and washed with 5% HCl solution (3 × 5 mL). The concentrated
organic layer was directly purified by silica gel column chromatography.
The product and byproducts a and b were
purified by elution with specific eluents. In path a, the recovered b was chlorinated with thionyl chloride by heating at 110–114
°C in toluene for 3 h and mixed with the recovered oxyma (a) in the presence of DIPEA and I was obtained
with 52% yield. In path b, we recovered I by recombination
of byproducts a and b in the presence of
silica gel under microwave irradiation with 35% yield. The yield of
recyclable coupling reagent was calculated with respect to the initial
amount of I used in the reaction. By this way, we were
able to recover byproducts and recombine to regenerate the coupling
reagent (I) very easily.
Scheme 1
Scheme 2
Scheme 3
Figure 1
Figure 2
Figure 3
Scheme 4
Scheme 5