Ethyl 2-cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o -NosylOXY): A recyclable coupling reagent for racemization-free synthesis of peptide, amide, hydroxamate, and ester

Article abstract of DOI:10.1021/jo500292m

Ubiquitousness of amide and ester functionality makes coupling reactions extremely important. Although numerous coupling reagents are available, methods of preparation of the common and efficient reagents are cumbersome. Those reagents generate a substantial amount of chemical waste and lack recyclability. Ethyl 2-cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o-NosylOXY), the first member of a new generation of coupling reagents, produces byproducts that can be easily recovered and reused for the synthesis of the same reagent, making the method more environmentally friendly and cost-effective. The synthesis of amides, hydroxamates, peptides, and esters using this reagent is described. The synthesis of the difficult sequences, for example, the islet amyloid polypeptide (22-27) fragment (with a C-terminal Gly, H-Asn-Phe-Gly-Ala-Ile-Leu-Gly-NH ₂) and acyl carrier protein (65-74) fragment (H-Val-Gln-Ala-Ala-Ile- Asp-Tyr-Ile-Asn-Gly-OH), following the solid-phase peptide synthesis (SPPS) protocol and Amyloid β (39-42) peptide (Boc-Val-Val-IIe-Ala-OMe), following solution-phase strategy is demonstrated. Remarkable improvement is noticed with respect to reaction time, yield, and retention of stereochemistry. A mechanistic investigation and recyclability are also described.

Full text of DOI:10.1021/jo500292m

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Article
Ethyl 2-cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o-
NosylOXY): A Recyclable Coupling Reagent for Racemization
free Synthesis of Peptide, Amide, Hydroxamate and Ester
Dharm Dev, Nani Babu Palakurthy, Kishore Thalluri, Jyoti Chandra, and Bhubaneswar Mandal
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo500292m • Publication Date (Web): 21 May 2014
Downloaded from http://pubs.acs.org on May 25, 2014
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Ethyl 2-cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o-
NosylOXY): A Recyclable Coupling Reagent for Racemization free
Synthesis of Peptide, Amide, Hydroxamate and Ester
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Dharm Dev, Nani Babu Palakurthy, Kishore Thalluri, Jyoti Chandra, and Bhubaneswar Mandal*
Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati. Assam.
India-781039
bmandal@iitg.ernet.in
ABSTRACT: Ubiquitousness of amide and ester functionality makes coupling reactions
extremely important. Although numerous coupling reagents are available, methods of
preparation of the common and efficient reagents are cumbersome. Those reagents generate
substantial amount of chemical waste and lack recyclability. Ethyl 2-cyano-2-(2-
nitrobenzenesulfonyloxyimino)acetate (o-NosylOXY), the first member of a new generation of
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coupling reagents, is found for which byproducts can be easily recovered and reused for the
synthesis of the same reagent making the method more environment friendly and cost effective.
Synthesis of amides, hydroxamates, peptides, and esters using this reagent is described.
Synthesis of the difficult sequences, for example, islet amyloid polypeptide (22-27) fragment
(with a C-terminal Gly, H-Asn-Phe-Gly-Ala-Ile-Leu-Gly-NH2) and acyl carrier protein (65-74)
fragment (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH), following solid phase peptide
synthesis (SPPS) protocol and Amyloid β (39-42) peptide (Boc-Val-Val-IIe-Ala-OMe) following
solution phase strategy is demonstrated. The remarkable improvement is noticed with respect to
reaction time, yield, and retention of stereochemistry. A mechanistic investigation and
recyclability are also described.
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INTRODUCTION
Amide and ester functionality are present in most of the natural products as well as
pharmaceutical compounds.1 Therefore, coupling reactions, which comprise activation of
carboxylic acids into an activated form that undergo acylation to produce esters, amides, and
peptides, are extremely important for academia and industry. Numerous coupling reagents have
been developed and commercialized including carbodiimides, phosphonium, and uronium/
aminium salts.2 Most of them engage the benzotriazole or azabenzotriazole ring system as an
important fragment of their structure, which is subsequently transformed into a good leaving
group.3 After two decades of domination of benzotriazole-based chemistry, with the pioneering
work of Albericio et al., a new class of peptide coupling reagents, that is, ethyl 2-cyano-2-
(hydroxyimino)acetate (Oxyma) based reagents, have been introduced.4 Oxyma is superior to its
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counterparts as racemisation suppressant and environment friendly reagent.5 Nevertheless,
existing popular coupling reagents (HOBt based coupling reagents as well as Oxyma based
coupling reagents) still suffer from three major drawbacks: (a) They generate lot of chemical
waste. For example, carcinogenic hexamethylphosphoramide (HMPA) and explosive
hydroxybezotriazole (HOBt) are generated as by-products, when benzotriazol-1-
yloxytris(dimethylamino)phos-phoniumhexafluorophosphate (BOP) is used as coupling reagent.6
Similarly, a urea derivative as well as HOBt is generated when N-(benzotriazol-1-yl)-1,1,3,3-
tetramethyluroniumhexafluoro phosphate (HBTU) is used.7 (b) Preparation protocols of these
reagents involve harsh conditions and toxic reagents. For example, HBTU is synthesized using
phosgene (inflammable, causes skin damage) and HOBt (explosive) in carbon tetrachloride, a
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carcinogenic
solvent.7
Similarly,
1-[(1-(cyano-2-ethoxy-2-oxoethylideneamino-oxy)-
dimethylamino-morpholinomethylene)] methanaminium hexafluorophosphate (COMU), an
Oxyma based coupling reagent, is also synthesized using phosgene. (c) Furthermore, recycling
those reagents may be cumbersome. Thus, they are not environment friendly and cost effective.
Therefore, invention of an ideal coupling reagent with highest level of efficacy but devoid of
epimerization tendency at the same time environment friendly and recyclable still remains a
challenge. We described herein a coupling reagent, Ethyl 2-cyano-2-(2-nitrobenzene
sulfonyloxyimino)acetate (o-NosylOXY, I), which have similar efficiency but devoid of the said
drawbacks.
Figure 1. Ethyl 2-cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o-NosylOXY, I)
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RESULTS AND DISCUSSION
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The reagent I, o-NosylOXY, was synthesized easily by the reaction between ortho-
nitrobenzenesulfonyl chloride and Oxyma in presence of diisopropylethylamine (DIPEA) under
nitrogen atmosphere.8 Reagent I is stable at room temperature (25 °C) and can be stored for long
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time. A time dependent HPLC and HNMR studies of I indicated no change until 20 days
(Supporting Information, Figure S112-S117). Its coupling efficiency was investigated using the
reaction between phenyl acetic acid and benzyl amine, DCM as a solvent (Scheme 1). Very good
yield (91 %) of the product, N-benzyl-2-phenylacetamide, was obtained in 2 h at room
temperature. Sulfonamide formation is an expected side reaction for such reagents. We
performed a model reaction between phenylacetic acid and benzyl amine using o-NosylOXY in
presence of DIPEA in DCM without pre-activation. After completion of the reaction, we found
40 % yield of N-benzyl-2-nitrobenzenesulfonamide and 50 % yield of N-benzyl-2-
phenylacetamide. To avoid such sulfonamide formation, we used 3-5 min pre-activation.
Scheme 1. Synthesis of amides and peptides using reagent I.
For solvent optimization we screened several solvents for the coupling reaction between phenyl
acetic acid and benzyl amine employing reagent I, and DCM was found to be the best solvent
(Table 1). Therefore, DCM was used for amidation, esterification and hydroxamate synthesis.
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Table 1. Solvent screening experiments
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aPerformed with phenylacetic acid (136 mg 1 equiv), Reagent I (1 equiv), DIPEA (2.2 equiv), benzyl amine (1
equiv), room temperature (25 °C). bYields refer to the isolated yield after column chromatography.
For racemization studies, we synthesized Z-DL-Phe-L-Ala-OMe and Z-L-Phe-L-Ala-OMe
using I and compared their HPLC profiles (Figure 2) to estimate the amount of racemization
caused by the coupling reagent. Appearance of the twin peak in the HPLC profile of Z-DL-Phe-
L-Ala-OMe corresponds to the two diastereomeric products, whereas, presence of the single
peak in that of Z-L-Phe-L-Ala-OMe demonstrates virtually no racemization occurred during the
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synthesis. Comparison of the H and 13C NMR spectra of these dipeptides also supports the
hypothesis (supporting information, Figure S29-S32). We found two doublets in 1H NMR at δ =
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1.32 and 1.21 ppm and similarly four peaks at δ = 173.0, 172.9, 170.9 and 170.7 ppm in C
NMR of Z-DL-Phe-L-Ala-OMe, which indicate the presence of two diastereomers. Whereas, we
found one doublet at δ = 1.32 ppm in the 1H NMR and two peaks at δ = 173.0 and 170.7 ppm in
13C NMR for Z-L-Phe-L-Ala-OMe indicating the presence of one diastereomer.
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Figure 2. Comparison of the HPLC profiles of Z-DL-Phe-L-Ala-OMe (left panel) and Z-L-Phe-
L-Ala-OMe (right panel) (C18 reverse phase analytical column, 5 µm, 25 cm× 4.6 mm, linear
gradient of 0-80 %, 0-7 min. then 80-100 % upto 20 min., CH3CN in H2O with 0.1% formic
acid)
However, supression of epimerization is known to occur for the presence of the N-terminal
urethane protecting group.9 To remove that doubt, we synthesized a tripeptide, Z-Gly-Phe-Val-
OMe (entry 18, Table 3), via coupling of Z-Gly-Phe-OH and H-Val-OMe in solution using I,
estimated the amount of racemization and compared that with reported data obtained for the
synthesis of the same tripeptide using other coupling reagents, such as, HBTU (N-[(1H-
benzotriazol-1-yl)(dimethylamino) methylene] N-methylmethanaminiumhexafluorophosphateN-
oxide),
methylmethanaminium
((dimethylamino)(morpholino)methylene)-1H-benzotriazoliumhexafluorophosphate-3-oxide)
HATU
(N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene)-N-
hexafluorophosphate-N-oxide), HDMB (1-
and
HDMA
(1-((dimethylamino)-(morpholino)methylene)-1H
[1,2,3]triazolo[4,5-
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b]pyridiniumhexafluorophosphate3-oxide).10 No racemization could be observed, when reagent I
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was used, however, epimerization was noted for other reagents (Table 2).
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Table 2. Comparison of yield and racemization tendency for the formation of Z-Gly-Phe-Val-
OMe using o-NosylOXY (I) with the reported10 results using various coupling reagents.
aNo racemization could be detected by the 1H and 13C NMR spectra and the HPLC profile (Supporting Information,
Figures S39- S40 and S128) within the limit of our experimental condition.
With the suitable conditions in hand, applicability of this method was explored with various
carboxylic acids and amines (Table 3). Interestingly, the reaction worked well with aromatic and
aliphatic carboxylic acids (entry 1-4, Table 3) as well as amino acids (entry 5-18). Even in case
of less nucleophilic amine, aniline (entry 4), the yield was found to be remarkably good.
However, repeated attempt of the reaction with 4-nitroaniline failed. It tolerates common amine
protecting groups including Fmoc and Cbz. Furthermore, the reaction worked well for sterically
hindered amino acids, e.g. Val (entries 10) and Aib (entries 16 and 17), as carboxylic acid
component as well as tertiary butyl amine (entry 6), Aib (entry 7), and Val (entry 18) as amine
component, which are known to be difficult for coupling. In all the cases, the yields were
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excellent and comparable with other reported common coupling reagents11 with no observed
racemization.
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We also performed the reaction between Fmoc-Phe-OH and NH2-Ala-OMe in the presence of o-
nitrobenzenesulfonyl chloride as a coupling reagent. In this case, we obtained dipeptide, Fmoc-
Phe-Ala-OMe, with 65 % yield and sulfonamide, o-nitrobenzenesulfonamide of methyl ester of
alanine, with 10% yield. About 10 % racemization was observed in the HPLC profile of the
dipeptide (Supporting Information, Figure S130-S132). This demonstrates the utility of the
Oxyma part in reagent I.
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Table 3. Synthesis of various amides and peptides under optimized condition using reagent I.
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aPerformed with Acid (1 equiv), Reagent I (1 equiv), DIPEA (2.2 equiv), Amine (1 equiv), room temperature (25
°C), 2-3 h. bYields were referred to the isolated yields after column chromatography.
Hydroxamates of amino acids are another interesting class of compounds which possess a
broad spectrum of medicinal properties.12 Therefore, we wanted to adopt the present
methodology for the synthesis of such important drug candidates.
Table 4. Synthesis of hydroxamates using I
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aPerformed with Acid (1 equiv), Reagent I (1 equiv), DIPEA (2.2 equiv), O-benzylhydroxylamine (1 equiv), room
temperature (25 °C), 2-3 h. bYields were referred to the isolated yields after column chromatography.
Our intial trials of coupling with model substrate, i.e. benzoic acid with O-benzyl hydroxyl
amine using reagent I yielded the desired product in very good yield (91%, entry 1, Table 4).
Then it was further extended to a variety of amino acids with varying side chain complexity
(entry 4-8, Table 4). Very good yields (81 to 92 %) were obtained on stirring at room
temperature (25 oC) for 2-3 hours.
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(a)
(b)
(c)
Figure 3. Sequences of peptides synthesised: (a) NFGAILG and (b) VQAAIDYING using SPPS,
HPLC and ESI-MS spectra (see supporting information, Figure S167-168); (c) Boc-VVIA-OMe
in solution.
We further envisaged to use reagent I for long chain peptide synthesis. As target, we took
IAPP (22-27) peptide (IAPP, Islet Amyloid Poly Peptide: H-Asn-Phe-Gly-Ala-Ile-Leu-Gly-NH2,
Figure 3a)13 and ACP (65-74) peptide fragment (ACP, Acyl Carrier Protein: H-Val-Gln-Ala-Ala-
Ile-Asp-Tyr-Ile-Asn-Gly-OH, Figure 3b).11 Both of them are known as difficult sequences for
synthesis as they contain many hydrophobic amino acids and undergo conformational change
resulting in aggregation during the synthesis.
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We achieved the synthesis of these peptides in very good yield (40% for the IAPP (22-27) and
50 % for the ACP (65-74) fragment after chromatographic purification, with respect to resin
loading) by stepwise coupling of constituent amino acids on rink amide MBHA resin using I as a
coupling reagent following Fmoc/tBu orthogonal protection technique based SPPS (Solid Phase
Peptide Synthesis) method. 3.2 fold excess of the Fmoc amino acids and 3 fold excess of the
coupling reagents were used for each coupling step and gently rotated. Care should be taken not
to take more amount of the coupling reagent than that of the N-protected amino acid, unlike other
coupling reagents, for avoiding truncation by sulfonamide formation. Although reagent I
generates higher yield in DCM than in DMF (Table 1), we used DMF as solvent for SPPS, as
DMF is more commonly used in SPPS. Couplings were accomplished smoothly in 2 hours,
which were confirmed by Kaiser test. A small amount of resin was taken after each coupling and
cleaved the peptide from resin using TFA/DCM/H2O mixture, precipitated using cold ether and
injected in analytical HPLC for examining the purity at each step. Each time single peak was
observed; eluent was collected and injected in ESI-MS for confirming the mass of the peptide
fragment. The HPLC retention times and ESI-MS data for each fragment during the synthesis of
IAPP (22-27) are mentioned in Table S1 in Supporting Information. The HPLC profiles and ESI-
MS spectra of the purified peptides are also provided in supporting information (Figure S153 and
154). Quality of the crude peptide also was excellent. As an example, that for peptide ACP (65-
74) was compared with the reported data (Table 5).11 Quality of the crude IAPP (22-27) peptide
fragment synthesized using I as coupling reagent was also very good (Supporting Information,
Figure S151). We also synthesized Boc-Val-Val-IIe-Ala-OMe, the C-terminal fragment of the
Amyloid β peptide.14 This is also a hydrophobic difficult sequence bearing huge steric
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hinderance (Figure 3c). A Boc-chemistry based stepwise coupling was performed in DCM
following solution phase methodology. Yield was good (78%, after five steps with respect to
starting Boc-Ile-OH) and HPLC chromatogram of the crude peptide (Supporting Information,
Figure S137) was as good as a purified peptide.
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Table 5. Comparative study of the purity of crude ACP (65-74) by TBTU, HATU, TBCR and o-
NosylOXY
a90% purity of the crude peptide could be detected by HPLC (supporting information Figure S165).
Soon completion of the establishment of the applicability of the coupling reagent I for the
synthesis of amides, hydroxamates and peptides, we investigated its efficiency for esterification
(Scheme 2). In spite of the numerous methodologies for the coupling reaction of alcohols and
carboxylic acids, it is still desired to develop an efficient coupling reagent for the said
transformation that is racemization free, with high yields for complicated substrates, clean
isolation of the products and most importantly, recyclable. Esterification using I addressed most
of these difficulties that were associated with the rest of the methodologies.
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Scheme 2. Synthesis of ester under optimized condition
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As a model reaction, we took phenyl acetic acid (1 mmol), in dichloromethane and added
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diisopropyl ethyl amine (1.1 mmol) to it. o-NosylOXY (1 mmol) was added to this mixture at
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room temperature (25 C) with stirring. After 5 min, benzyl alcohol (1 mmol) and DIPEA (1.1
mmol) was added. After 2 h, the reaction was found to be complete and furnished the desired
product in very good yield (92 %).
Table 6. Scope of esterification using reagent I
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aPerformed with Acid (1 equiv), Reagent I (1 equiv), DIPEA (2.2 equiv), Alcohol (1 equiv), room temperature (25
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°C), 2-3.5 h. bYields refer to the isolated yields after column chromatography.
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After standardization of the reaction conditions, we investigated the generality of the present
protocol applying it to various alcohols and carboxylic acids. The reaction of phenyl acetic acid
with varieties of aromatic (entry 1-4, Table 6) and aliphatic (entry 5-7) alcohols worked well.
From the observation of the yields of entries 1-4, it was evident that the presence of electron
donating group on phenol retards the reaction and thereby yields lesser product than its
counterparts, which is in agreement with the stability of the phenoxide ions. The reaction went
well with secondary (entry 7) and tertiary alcohol (entry 6).
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The present protocol was successfully applied for the synthesis of biologically active esters
also. The aliphatic carboxylic acids (entry 1-3, Table 7) worked well. The mentioned
electromeric affect holds true for these reactions as well. In case of salicylic acid, i.e. entry 4, the
reaction was chemoselctive and produced 4-nitro-benzyl ester of salicylic acid as a major
product; although two nucleophilic attacks were logically possible. Self-condensation was very
less due to the lesser nuclephilicity of the hydroxyl group of the salicylic acid than that of the p-
nitrobenzyl alcohol. Phenolic acids have been esterified with excellent chemoselectivity in
presence of strong protic acids (Fischer esterification), but harsh reaction conditions and excess
of alcohol was required to drive the reaction to a satisfactory degree of conversion,15 whereas, it
worked smoothly in the present protocol, which is milder. Furthermore, in case of entry 5 and 6
(Table 7) the product observed was the ester alone, not the corresponding amide. Various
aliphatic primary alcohols (entries 4-11, Table 7) and a secondary alcohol (entry 7) were
subjected to the reaction conditions and the yield was good (82-90 %).
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Table 7. Synthesis of various carboxylic esters using I.
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aPerformed with Acid (1 equiv), Reagent I (1 equiv), DIPEA (2.2 equiv), Alcohol (1 equiv), room temperature (25
°C). bYields refer to the isolated yields after column chromatography.
Even amino acids (entry 9-11, Table 7) retained the same reactivity towards this reaction. In
order to estimate the extent of racemization, we initially synthesized Boc-DL-Phe-OBn using our
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protocol and passed through a chiral column. Two distinct peaks, corresponding to the two
enantiomers, were observed in HPLC chromatogram (Figure 4, Left panel). Next, we synthesized
Boc-L-Phe-OBn following the same strategy. Chromatographic signature of Boc-L-Phe-OBn
reveals only one peak corresponding to the enantiopure product (Figure 4, Right panel).
Therefore, it was inferred that the present protocol does not cause any detectable racemization.
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Figure 4. HPLC chromatogram of Boc-D,L-Phe-OBn (Left panel) and Boc-L-Phe-OBn (Right
panel, CHIRAL PAKR AS-H column, 5 µm, 2.1×150 mm, an isocratic gradient of 10%
isopropanol in hexane was used).
Scheme 3. Plausible pathways for the condensation
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A plausible mechanism is depicted in Scheme 3. Initially, the nucleophile generated by the
deprotonation of the carboxylic acid in presence of DIPEA attacks the sulfonyl centre of I.
Expulsion of the resonance stabilized Oxyma anion II results in the mixed anhydride III. Further
nucleophilic attack of II on the carbonyl carbon of III and expulsion of the sulfonic acid IV
results in the formation of the intermediate V, the activated Oxyma ester of the carboxylic acid.
V undergoes nucelophilic substitution to produce the corresponding ester or amide. Isolation and
complete characterization using 1H NMR and 13C NMR (Supporting Information, Figure S1-S2)
and XRD analysis of the intermediate V was achieved (Supporting Information, Figure S169).
However, the added nucleophile (amine or alcohol) may also react with the intermediate III. But,
in that case, sulfonamide also may get generated as byproduct and racemization of the product is
expected, as it was observed when the reaction was performed without pre-activation or using o-
nitrobenzenesulfonyl chloride. A 3-5 min pre-activation precludes that possibility by increasing
the population of the intermediate V in the reaction mixture.
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The product as well as by-products Oxyma (II) and 2-nitrobenzene sulfonic acid (IV) were
recovered easily. After completion of the reaction (entry 2, Table 4), the reaction mixture was
diluted with ethyl acetate and washed with 5% citric acid solution (3×5 ml). Concentrated
organic layer was directly purified by a silica gel column chromatography. The product and
byproducts were eluted with specific eluents. Purity of the recovered II and IV was as good as
the commercial samples (supporting information Figure S103-S105). The recovered sulfonic
acid was chlorinated by heating on 60 oC for 1.5 h in toluene with a small amount of DMF using
thionyl chloride. Pure sulfonyl chloride, obtained in this way, was transformed to I by stirring
with Oxyma in basic medium (DIPEA).
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Scheme 4. Recyclability of coupling reagent I. aYield of the regenerated I from the recovered II
and IV was calculated with respect to the initial amount of I used in the reaction.
To the best of our knowledge, only one such recyclable coupling reagent is reported, which is
a hypervalent iodine-III reagent, iodosodilactone, and it works in association with DMAP and
PPh3.16 Dipeptide formation with this combination of reagents usually takes 8 h under heating
condition (60 °C). On the other hand, it takes only 1 h at room temperature (25 °C) by our
method. Moreover, stoichiometric amount of triphenyl phosphine oxide is generated with
iodosodilactone/DMAP/PPh3, whereas the present method does not leave any solid waste to the
environment.
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CONCLUSION
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We have introduced a new and efficient coupling reagent, o-NosylOXY (I), for the synthesis
of amide, hydroxamate, peptide and ester. The present method is associated with the following
important features: 1) apparently complete retention of stereochemistry, 2) applicability to both
solution and solid phase synthesis of large peptides with difficult sequences, 3) ease of
preparation of the reagent, 4) works under ambient and milder conditions. A plaussible
mechanism is suggested and an intermediate could be isolated and fully characterized which
supports that. Most importantly, the reagent is recyclable, therefore, cost effective and
environment friendly. All these features makes this reagent useful for industry and academia in
the context of condensation chemistry.
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EXPERIMENTAL SYSTEM
General consideration:
All reagents were purchased from commercial sources and were used without any further
purification unless mentioned otherwise. o-nitrobenzenesulfonyl chloride is freshly recrystallized
before use. Dichloromethane is distilled and dried using standard procedure. Melting points were
uncorrected. Thin layer chromatograms were run on glass plates coated with silica gel G for
TLC, using solvent systems EtOAc/Hexane. Compounds were purified by column
chromatography using Silica Gel (60-120 mesh) with EtOAc/Hexane (of specific proportion as
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required) as an eluent. H NMR (400 MHz and 600 MHz) and 13C NMR (100 MHz and 150
MHz) were recorded using CDCl3 as solvent. Chemical shifts (δ) are reported in parts per million
(ppm), internal reference (0.05% to 1 %) tetramethylsilane. Coupling constants (J) are reported
in Hz singlet (s), doublet (d), triplet (t), doublet of doublet (dd), multiplet (m), or broad (br).
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High resolution mass spectra were recorded on a Q-TOF ESI-MS instrument and a Q-TOF
LC/MS system; HPLC analysis was carried out with either a C8 (5 µm, 3.5 × 150 mm) or a C18
(5 µm, 4.6 × 250 mm) reverse phase column and a chiral column (5 µm, 2.1×150 mm) coupled
to a UV detector. HPLC grade solvents were used for HPLC analysis. IR spectra were recorded
on a IR spectrometer; X-ray data were collected on a diffractometer equipped with a CCD area
detector using Mo. Data for previously reported compounds (cited) matched well with our
observed data.
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Procedure for Synthesis of Coupling Reagent Ethyl 2-cyano-2-(2-nitrobenzenesulfonyloxy
imino) acetate (o-NosylOXY, I):
DIPEA (0.87 ml, 5 mmol, 1 equiv) was added to a solution of Oxyma (710 mg, 5 mmol, 1
equiv) in 2 ml of DCM under nitrogen. The reaction mixture was cooled up to 0 oC and followed
by dropwise addition of 2-nitrobenzenesulfonyl chloride (1108 mg, 1 mmol, 1 equiv) for 30 min.
The reaction mixture was stirred for another 2 h at room temperature (25 °C). After completion
of the reaction, the reaction mixture was diluted with 10 ml of DCM and washed with 5% of HCl
(3×5 ml). Finally organic layer dried by anhydrous CaCl2 and recrystallized with hexane. Rf =
1
0.50 (EtOAc:Hexane, 1:4), Yield 1308 mg, 80 %; colorless crystalline solid; H NMR (400
MHz, CDCl3): δ 8.26-8.24 (d, J = 8.0 Hz, 1H), 7.95-7.90 (m, 1H), 7.89-7.84 (m, 2H), 4.45-4.40
(q, 2H), 1.39-1.36 (t, J = 8.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 155.5, 148.6, 137.3,
133.3, 132.9, 132.7, 126.7, 125.5, 105.9, 65.0, 13.8; IR (KBr) 3103, 2988, 2224, 1748, 1544.89,
928, 742 cm-1; HRMS (ESI) m/z: [M+H]+ calcd for C11H10N3O7S 328.0239, found 328.0235.
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General Procedure for the Synthesis of Ester and Amide:
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o-NosylOXY (1 equiv) was added to a solution of acid (1 equiv) and DIPEA (1 equiv) in 2
ml of DCM. The reaction mixture was stirred for 3-5 min. for preactivation followed by the
addition of alcohol or amine (1.0 equiv). The reaction mixture was stirred at room temperature
for 2-3 h. After completion of the reaction, the reaction mixture was diluted with 50 ml of ethyl
acetate; the organic phase was washed with 5% citric acid (3×20 ml), 5% NaHCO3 (3×20 ml)
and dried by anhydrous Na2SO4. Finally, Na2SO4 was filtered off and the solvent was evaporated
to obtain the product which was purified by column chromatography.
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General Procedure for the Synthesis of Dipeptides:
To a solution of N-protected amino acid (1 equiv) and o-NosylOXY (1 equiv) in 2 ml of
DCM, DIPEA (1.1 equiv) was added. The reaction mixture was stirred for 5 min. for
preactivation followed by the addition of methyl ester of the second amino acid (1.1 equiv) and
DIPEA (1.1 equiv) in 1 ml of DCM. The reaction mixture was stirred at room temperature for
more 2-3 h. After completion of the reaction, the reaction mixture was diluted with 50 ml of
ethyl acetate, the organic phase was washed with 5% citric acid (3×20 ml), 5% NaHCO3 (3×20
ml), brine and dried over anhydrous Na2SO4. Finally, Na2SO4 was filtered and the solvent was
evaporated. The product was purified by silica gel column chromatography.
General Procedure for the Synthesis of Hydroxamate:
To a solution of acid or N-protected amino acid (1 equiv) and o-NosylOXY (1 equiv) in 2 ml
of DCM, DIPEA (1.1 equiv) was added. The reaction mixture was stirred for 3-5 min. for
preactivation followed by the addition of O-benzylhydroxylamine (1.1 equiv) and DIPEA (1.1
equiv) in 1 ml of DCM. The reaction mixture was stirred at room temperature (25 °C) for more
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2-3 h. After completion of the reaction, the reaction mixture was diluted with 50 ml of ethyl
acetate, the organic phase was washed with 5% citric acid (3×20 ml), 5% NaHCO3 (3×20 ml),
brine and dried over Na2SO4 anhydrous. Finally, Na2SO4 was filtered and the solvent was
evaporated. The product was purified by silica gel column chromatography.
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Solution Phase Synthesis of Boc-VVIA-OMe:
Boc-isoleucine (750 mg, 3.24 mmol, 1 equiv,) and o-NosylOXY (1059 mg, 3.24 mmol, 1
equiv) was taken in a 25 ml RB, after that DIPEA (0.85 ml, 4.86 mmol, 1.5 equiv) was added
and kept 5 min for pre-activation. In another oven dried 50 ml RB, methyl ester of alanine (675
mg, 4.86 mmol, 1.5 equiv) was taken in DCM and DIPEA was added to it until basic pH was
reached. Finally, this solution was added in first RB and stirring continued until completion of
the reaction. Then, the reaction mixture was diluted by 50 ml of EtOAc, washed by 5% NaHCO3
solution (2 × 5 ml) and washed by 5% citric acid solution (2 × 5 ml). Finally, combined organic
layer was dried using anhydrous Na2SO4. Solid product (Boc-IA-OMe) was obtained after
evaporation of EtOAc by rotary vacuum evaporator.
In an oven dried 50 ml RB, Boc-IA-OMe was taken and 70% TFA solution in DCM was added
to it. Starring continued up to 2.5 h. After that, TFA was evaporated by rotary evaporator and
washed 3 times with diethyl ether and finally white solid (IA-OMe) was obtained.
After Boc deprotection, resulted IA-OMe was coupled with Boc-V-OH following the above
mentioned procedure to obtain Boc-VIA-OMe. Another cycle of Boc-deprotection and coupling
with Boc-V-OH, resulted white solid Boc-VVIA-OMe, which was characterized by reverse
phase HPLC, retention time was 12.8 minutes on linear gradient of 20 to 100% CH3CN/0.09%
TFA in H2O/0.09% TFA over 18 min, symmetry C8 analytical column and LRMS (ESI): m/z
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[M+H]+ calculated for C25H47N4O7 is 537.3264 found 537.3421. Yield was 1298 mg, 78% with
respect to starting material Boc-isoleucine.
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Solid Phase Synthesis of NFGAILG-NH2 and VQAAIDYING-NH2:
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Specific amino acids were manually assembled on Fmoc-Rink Amide MBHA resin (loading
1.1 mmol/g) following Fmoc-tBut orthogonal protection strategy. Fmoc-amino acids (3.2 equiv),
o-NosylOXY (3 equiv) and DIPEA (5 equiv) was kept for pre-activation for 5 min. Amino acid
coupling was performed for 2 h. Fmoc deprotection was carried out with 25% piperidine/DMF (3
× 7 min). The peptide was cleaved from the resin using TFA/DCM/H2O mixture for 2 h 30 min.
Purification of the peptide was performed using semi-preparative HPLC using a linear gradient
of 20 min (5 to 100 % ACN in water with 0.09% TFA) and characterized using ESI-MS.
For NFGAILG-NH2, we took 300 mg of Fmoc-Rink Amide resin (loading 1.1 mmol/g), after
final coupling, peptide was cleaved from resin and purified by semi-preparative HPLC. The
purified yield of peptide was 90 mg, 40 %, HPLC profile and ESI-MS spectrum are shown in
supporting information (S153-154).
And for VQAAIDYING-NH2, we took 300 mg of Fmoc-Rink Amide resin (loading 1.1
mmol/g), after final coupling, this peptide was also cleaved from resin and purified by semi-
preparative HPLC. Yield of the purified peptide was 175 mg, 50 %. HPLC profile and ESI-MS
spectrum are provided in supporting information (S167-168).
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Intermediate V (Scheme 3): Oxyma ester naphthanoic acid
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White solid; Rf = 0.40 (EtOAc:Hexane, 1.0:9.0), H NMR (600 MHz, CDCl3) δ 8.72 (s, 1H),
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8.13-8.11 (t, J = 8.0 Hz, 1H), 8.00-7.98 (d, J = 12.0, Hz, 1H), 7.64-7.61 (t, J = 12.0 Hz, 2H),
7.64-7.61 (t, J = 6.5, Hz, 1H), 7.58-7.56 (t, J = 6.6, Hz, 1H), 4.46-4.43 (q, J = 6.0 Hz, 2H), 1.43-
1.40 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 160.9, 157.1, 136.5, 133.1, 132.4,
129.9, 129.7, 129.2, 128.0, 127.4, 125.0, 122.8, 107.2, 64.7, 14.1; IR (KBr): 2924, 2853, 2224,
1715, 1646, 1593, 1541, 1398, 668, 544 cm-1; HRMS (ESI) m/z: [M+Na]+ calcd for
C16H12N2NaO4 319.0695, found 319.0691. CCDC-936483 contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
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Methyl
2-(2-((((9H-fluoren-9-yl)methoxy)
carbonyl)amino)-3-phenylpropanamido)
propanoate17 (by o-nitrobenzenesulfonyl chloride). Yield: 307 mg (65%); white solid; Rf =
1
0.40 (EtOAc:Hexane, 3.0:7.0); m.p.178-180 °C; H NMR (400 MHz, CDCl3): δ 8.09-8.07 (m,
1H), 7.94-7.91 (m, 1H), 7.77-7.72 (m, 2H), 7.55-7.52 (t, J = 12.0 Hz, 2H), 7.42-7.38 (t, J = 12.0
Hz, 2H), 7.32-7.24 (m, 4H), 7.21-7.19 (m, 1H), 6.33 (br, 1H), 5.39 (br, 1H), 4.52-4.43 (m, 3H),
4.32-4.23 (m, 2H), 4.20-4.17 (t, J = 8.0 Hz, 1H), 3.52 (s, 3H), 3.12-3.05 (m, 1H), 1.50-1.48 (d, J
= 8.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 172.9, 172.1, 170,67, 156.08, 143.9, 141.4,
136.4, 133.7, 132.9, 130.6, 129.5, 128.5, 127.8, 127.8, 127.2, 125.7, 120.1, 67.2, 56.1, 52.2, 48.3,
47.2, 19.7, 18.3; IR (KBr): 3304, 1738, 1690, 1652, 1534, 1262, 698 cm-1; HRMS (ESI): m/z
[M+H]+ calcd for C28H29N2O5 473.2076, found 473.2079.
(L) Methyl 2-(2-nitrophenylsulfonamido)propanoate (by o-nitrobenzenesulfonyl
chloride)18: pale yellow solid; Rf = 0.50 (EtOAc:Hexane, 3.0:7.0); m.p.68-70 °C; 1H NMR (400
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MHz, CDCl3): δ 8.29-8.27 (m, 1H), 8.07-8.05 (m, 1H), 7.92-7.90 (m, 1H), 7.74-7.71 (m, 1H),
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6.18 (br, 1H), 4.23-4.19 (m, 1H), 3.50 (s, 3H), 1.50-1.48 (d, J = 8.0 Hz, 3H); C NMR (150
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MHz, CDCl3): δ 172.1, 147.9, 134.4, 133.8, 133.0, 130.6, 125.7, 52.7, 48.8, 16.7; IR (KBr):
3330, 2921, 1746, 1691, 1536, 1261, 1184, 741 cm-1; HRMS (ESI): m/z [M+Na]+ calcd for
C10H12N2NaO6S 311.0314, found 311.0317.
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N-benzyl-2-nitrobenzenesulfonamide (byproduct of the reaction without pre-activation):
pale yellow solid; Rf = 0.52 (EtOAc:Hexane, 3.0:7.0); 1H NMR (400 MHz, CDCl3): δ 8.01-7.98
(m, 1H), 7.83-7.81 (m, 1H), 7.70-7.63 (m, 2H), 7.31-7.22 (m, 5H), 5.77 (br, 1H), 4.33-4.32 (d, J
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= 4.0 Hz, 1H); C NMR (100 MHz, CDCl3): δ 147.9, 135.8, 134.1, 133.6, 132.9, 131.1, 128.8,
128.0, 125.4, 48.0; IR (KBr): 3340, 2961, 1691, 1535, 1221, 1182, 933, 742 cm-1; HRMS (ESI):
m/z [M+Na]+ calcd for C13H12N2NaO4S 315.0415, found 315.0420.
N-benzylbenzamide (entry 1, Table 3).19 Yield: 186 mg (88%); white solid; Rf = 0.50
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(EtOAc:Hexane, 2.0:8.0) mp: 102-104 °C. H NMR (400 MHz, CDCl3): δ 7.78-7.76 (m, 2H),
7.42-7.37 (m, 4H), 7.36-7.28 (m, 4H), 4.63-4.62 (d, J = 4.0 Hz, 2H); 13C NMR (100 MHz,
CDCl3): δ 167.6, 138.4, 134.4, 131.6, 128.8, 128.6, 127.9, 127.5, 127.1, 44.1; IR (KBr): 3290,
2924, 1637, 1551, 765, 565 cm-1; HRMS (ESI): m/z [M+H]+ calcd for C14H14NO 212.1075,
found 212.1073.
N-benzyl-2-phenylacetamide (entry 2, Table 3).19 Yield: 205 mg (91%); white solid; Rf = 0.51
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(EtOAc:Hexane, 2.0:8.0); mp: 76-78 °C. H NMR (400 MHz, CDCl3): δ 7.36-7.27 (m, 5H),
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7.24-7.16 (m, 5H), 5.76 (br, 1H), 4.42 (s, 2H), 3.62 (s, 2H); C NMR (100 MHz, CDCl3): δ
171.1, 138.3, 135.0, 129.4, 129.0, 128.7, 127.5, 127.4, 127.3, 43.7, 43.6; IR (KBr): 3289, 2925,
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1638, 1552, 784, 634, 566 cm-1; HRMS (ESI): m/z [M+H]+ calcd for C15H16NO 226.1232, found
226.1231.
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N-(4-methoxyphenyl)-2-phenylacetamide (entry 3, Table 3). Yield: 198 mg (82%); pale
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yellowish solid; Rf = 0.40 (EtOAc:Hexane, 3.0:7.0); m.p.121-123°C; H NMR (600 MHz,
CDCl3): δ 7.39-7.37 (m, 2H), 7.35-7.29 (m, 5H), 7.12 (br, 1H), 6.81-6.79 (d, J = 12.0 Hz, 2H),
3.75 (s, 3H), 3.70 (s, 2H); 13C NMR (150 MHz, CDCl3): δ 169.3 156.7, 134.7, 130.8, 129.7,
129.3, 127.7, 122.0, 114.2, 55.6, 44.8; IR (KBr): 3330, 2930, 2832, 1751, 1519, 1262, 1188,
1016, 544 cm-1; HRMS (ESI): m/z [M+H]+ calcd for C15H16NO2 242.1181, found 242.1183.
2-N-Diphenyl-acetamide (entry 4, Table 3).20 Yield: 181 mg (86%); white solid; Rf = 0.40
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(EtOAc:Hexane, 2.0:8.0); mp: 115-117 °C. H NMR (400 MHz, CDCl3): δ 7.42- 7.36 (m, 5H),
7.35-7.06 (m, 5H), 3.73 (s, 2H); 13C NMR (100 MHz, CDCl3): δ 169.8, 137.9, 134.7, 129.5,
129.1, 128.9, 127.5, 124.5, 120.2, 44.6; IR (KBr): 3285, 2967, 1657, 1601, 865, 543 cm-1;
HRMS (ESI): m/z [M+H]+ calcd for C14H14NO 212.1075, found 212.1071.
Benzyl 2-oxo-2-(piperidin-1-yl) ethylcarbamate (entry 5, Table 3).21 Yield: 265 mg (89%);
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white solid; Rf = 0.30 (EtOAc:Hexane, 4.0:6.0); mp: 113-116 °C; H NMR (400 MHz, CDCl3):
δ7.32-7.28 (m, 5H), 5.84 (br, 1H), 5.09 (s, 2H), 3.98-3.97 (d, J = 4.0 Hz, 2H), 3.54-3.52 (t, J =
5.2 Hz, 2H), 3.29-3.27 (t, J = 5.2 Hz, 2H), 1.70-1.68 (d, J = 4.0 Hz, 2H), 1.63-1.61 (d, J = 4.8 Hz,
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2H), 1.54-1.52 (d, J = 5.6 Hz, 2H); C NMR (100 MHz, CDCl3): δ 166.0, 156.3, 136.6, 128.5,
128.1, 128.0, 66.8, 45.4, 43.1, 42.6, 34.0, 26.2, 25.7, 25.4, 25.0, 24.3; IR (KBr): 3293, 2936,
1707, 1641, 763, 541 cm-1; HRMS (ESI): m/z [M+Na]+ calcd for C15H20N2NaO3 299.1372,
found 299.1368.
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(9H-fluoren-9-yl)methyl (1-(tert-butylamino)-1-oxopropan-2-yl) carbamate (entry 6, Table
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3). Yield: 296 mg (81%); yellowish solid; Rf = 0.40 (EtOAc:Hexane, 3.0:7.0); [α]D = + 4.07
(CHCl3, c = 0.54); 1H NMR (600 MHz, CDCl3): δ 7.76-7.75 (d, J = 12.0 Hz, 2H), 7.59-7.57 (d, J
= 12.0 Hz, 2H), 7.41-7.38 (m, 2H), 7.32-7.29 (m, 2H), 4.43-4.37 (m, 3H), 4.24-4.19 (t, J = 6.0
Hz, 1H), 4.13 (br, 1H), 1.36-1.35 (d, J = 6.0 Hz, 3H), 1.34 (s, 9H); 13C NMR (150 MHz, CDCl3):
δ 171.6, 156.1, 144.0, 141.5, 127.9, 127.2, 125.2, 120.1, 67.2 51.5, 47.3, 28.8, 19.1; IR (KBr):
3330, 3231, 2984, 1754, 1243, 752, 542 cm-1; HRMS (ESI): m/z [M+H]+ calcd for C22H27N2O3
367.2022, found 367.2025.
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(L) Methyl 2-(2-((((9H-fluoren-9-yl)methoxy) carbonyl)amino) propanamido)-2-
methylpropanoate (entry 7, Table 3). Yield: 328 mg (80%); white solid; Rf = 0.50
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(EtOAc:Hexane, 3.0:7.0); H NMR (400 MHz, CDCl3): δ 7.77-7.75 (d, J = 12.0 Hz, 2H), 7.62-
7.58 (m, 2H), 7.42-7.38 (t, J = 12.0 Hz, 2H), 7.32-7.29 (t, J = 12.0 Hz, 2H), 5.42 (br, 1H), 4.43-
4.37 (m, 2H), 4.24-4.21 (t, J = 8.0 Hz, 1H), 4.16-4.10 (m, 1H), 3.76 (s, 3H), 1.44-1.42 (d, J =
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12.0 Hz, 3H), 1.25 (s, 6H); C NMR (100 MHz, CDCl3): δ 173.7, 155.8, 144.1, 141.5, 127.9,
127.2, 125.2, 124.9, 120.2, 76.9, 67.2, 52.7, 50.5, 49.8, 47.3, 29.7, 18.9; IR (KBr): 3341, 1722,
1654, 1650, 1231, 664 cm-1; HRMS (ESI): m/z [M+H]+ calcd for C23H27N2O5 411.1920, found
411.1922.
Methyl 2-(2-(((9H-fluoren-9-yl) methoxy)carbonyl) acetamido)acetate (entry 8, Table 3).
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Yield: 321 mg (91%); white solid; Rf = 0.31 (EtOAc:Hexane, 4.0:6.0); mp: 162-163 °C; H
NMR (400 MHz, CDCl3): δ 7.76-7.74 (d, J= 8.0 Hz, 2H), 7.58-7.56 (d, J = 8.0 Hz, 2H), 7.40-
7.37 (t, J= 8.0 Hz, 2H), 7.31-7.27 (t, J = 8.0 Hz, 2H), 6.80 (br, 1H), 5.73 (br, 1H), 4.43-4.41 (d,
J = 6.8 Hz, 2H), 4.22-4.19 (t, J = 7.2 Hz, 1H), 4.04-4.03 (d, J = 8.0 Hz, 2H), 3.92-3.91 (d, J =
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6.8 Hz, 2H), 3.73 (s, 3H); C NMR (100 MHz, CDCl3): δ 169.9, 169.7, 156.8, 143.7, 141.2,
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