Fmoc-Amox, A Suitable Reagent for the Introduction of Fmoc

Article abstract of DOI:10.1021/acs.oprd.7b00199

Synthesis of most peptides is achieved using solid-phase peptide synthesis employing the Fmoc/tert-butyl strategy. However, the introduction of Fmoc in N-unprotected amino acids seems to be challenging due to the formation of dipeptides and sometimes tripeptides as impurities and β-alanyl impurities when Fmoc-OSu is used as well. Herein, we report an efficient and successful method using Fmoc-Amox, which is an oxime based derivative, toward the synthesis of Fmoc-glycine with no traces of side reactions. Fmoc-Amox is inexpensive, and Amox can be easily removed after the reaction, thus affording pure Fmoc-Gly-OH devoid of any detrimental impurities or contamination, mainly dipeptide or Amox itself, as shown by high-performance liquid chromatography and NMR, respectively.

Full text of DOI:10.1021/acs.oprd.7b00199

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Full Paper
Fmoc-Amox, a Suitable Reagent for the Introduction of Fmoc
Ashish Kumar, Anamika Sharma, Elvira Haimov, Ayman El-
Faham, Beatriz G. de la Torre, and Fernando Albericio
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00199 • Publication Date (Web): 14 Aug 2017
Downloaded from http://pubs.acs.org on August 15, 2017
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Fmoc-Amox, a Suitable Reagent for the Introduction of Fmoc
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Ashish Kumar,a,b Anamika Sharma,b Elvira Haimov,c Ayman ElꢀFahamd,e Beatriz G. de la
Torre,b,f* and Fernando Albericioa,b,e,g*
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aSchool of Chemistry and Physics, University of KwaZuluꢀNatal, University Road, Westville,
Durban 4001, South Africa
bCatalysis and Peptide Research Unit, School of Health Sciences, University of KwaZuluꢀNatal,
University Road, Westville, Durban 4001, South Africa.
cLuxembourg BioTechnologies, 8 Sapir Pinhas St., Nes Ziona 7403631, Israel
dDepartment of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
Alexandria 21321, Egypt.
eDepartment of Organic Chemistry, University of Barcelona, Martí i Franqués 1ꢀ11, 08028ꢀ
Barcelona, Spain.
fKRISP, College of Health Sciences, University of KwaZuluꢀNatal, Westville, Durban 4001,
South Africa.
gCIBERꢀBBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
Barcelona Science Park, Baldiri Reixac 10, 08028ꢀBarcelona, Spain
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KEYWORDS
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Solidꢀphase peptide synthesis, Fmoc, protecting groups, side reactions, dipeptide formation,
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βꢀalanine.
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ABSTRACT
Synthesis of most peptides is achieved using SPPS employing Fmoc/tBu strategy. However,
introduction of Fmoc in Nꢀunprotected amino acids seems to be challenging due to the formation
of dipeptides and sometimes tripeptides as impurities, and βꢀalanyl impurities when FmocꢀOSu
is used as well. Herein, we report an efficient and successful method using FmocꢀAmox, which
is an oxime based derivative, towards the synthesize of Fmocꢀglycine with no traces of side
reactions. FmocꢀAmox is inexpensive and Amox can be easily removed after the reaction, thus
affording pure FmocꢀGlyꢀOH devoid of any detrimental impurities or contamination, mainly
dipeptide or Amox itself, as shown by HPLC and NMR, respectively.
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In 1963, Merrifield described a whole new concept of chemical synthesis,1 with first of all great
impact in research, but that at the end has changed the pharmacopoea.2ꢀ3 Nowadays, there are
drugs in market whose active pharmaceutical ingredients (APIs) are TIDES (Oligonucleotides
and Peptides Therapeutics), containing up to 30ꢀ40 monomers,4 which have been produced using
the solidꢀphase methodology described first by Merrifield.1 Although at the beginning, the solidꢀ
phase peptide synthesis (SPPS) strategy was in some manner criticized by his European
colleagues,5 currently it has been totally adapted in research field as well as in the production.6ꢀ8
The idea is simple, as the name indicates, all amino acids have at least two functions, a
carboxylic acid and an amine, and therefore if the reactivity of carboxylic acid of the amino acid
Cꢀterminal is masked with an insoluble polymeric permanent group and the amine with a
temporary protecting groups, the stepwise synthesis is performed by sequential cycles which
involves removal of the protecting group from amine followed by coupling of the next Nꢀ
protected amino acid.1, 8ꢀ9 However, in case of trifunctional amino acids, side chain is protected
with a permanent (or semi permanent) protecting group has been employed.10ꢀ11 During the early
years, Merrifield used benzyl (Bzl) type groups for permanent protection and tertꢀ
butyloxycarbonyl (Boc) as temporary one.1, 11 However, Boc and Bzl are not orthogonal as they
both can be cleaved under acidolytic condition: trifluoroacetic acid (TFA) for Boc and stronger
acids such as HF or trifluoromethanesulfonic acid (TFMSA) for Bzl.12 In the earliest 70’s,
Carpino, who had also described the Boc group, revolutionized the area of peptide chemistry by
proposing the fluorenylmethoxycarbonyl (Fmoc) group as temporary Nꢀamino protection which
is removed in presence of bases and hence is orthogonal to tertꢀbutyl (tBu) group.13ꢀ14 In
addition, Sheppard and Atherton in Europe15 parallel to Chang and Meienhofer in USA16,
developed the soꢀcalled Fmoc/tBu strategy, which at the end liberates the peptide after treatment
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with TFA solution.17 The implementation of this methodology signified a democratization of the
peptide synthesis, because the use of Boc/Bzl strategy implied the concourse of wellꢀtrained
chemists and special equipment for the cleavage reaction, while with the Fmoc/tBu strategy,
other laboratories with more bio focus are also able to synthesize peptides.12 Furthermore, it also
allows the production of peptides in multiꢀkilogram scale.6ꢀ7
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At the beginning, commercial Fmocꢀamino acids were prepared by the reaction of amino acids
with FmocꢀCl in basic pH following SchottenꢀBaumann conditions (Figure 1A).13ꢀ14 However,
in the earlier 80’s, our group18 and Verlander at BioResearch, Inc., (later Bachem Americas) in
collaboration with Goodman19 described independently that most of the commercial Fmocꢀamino
acids contains dipeptide and even tripeptides. These impurities come from the high reactivity of
FmocꢀCl, which could also react with the carboxylic group of the amino acid to be protected
rendering an anhydride, which in turn could react with the amine terminus of another molecule
of amino acid leading to the formation of dipeptide according to the mechanism as shown in
Figure 1B.
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Figure 1: Fmoc protection of Amino acids: A. Mechanism of Fmoc protection. B. Mechanism
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for the formation of protected dipeptides during the protection of amino acids.
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Taking into consideration that even a small percentage of impurities can lead to decrease of the
final yield and purity, both groups came to a common conclusion of avoiding the use of Fmocꢀ
Cl. Since these sideꢀreactions are associated with the quality of the leaving group, we proposed
the use FmocꢀN3,18 which had been already proposed by Carpino in his seminal paper.13 and
Verlander, Goodman, et al suggested the use of Fmocꢀsuccinimidyl carbonate (FmocꢀOSu), after
screening different leaving groups.19 To avoid the preparation and storage of FmocꢀN3, our group
proposed in situ preparation of FmocꢀN3 from FmocꢀCl and NaN3. This strategy render Fmocꢀ
amino acids with high range of purity.20 Parallel to this, Bolin et al. proposed the use of FmocꢀCl
with an in situ protection of carboxyl group with silylating reagent.21 Years later, Barlos et al.
also proposed this method for the preparation of trityl (Trt)ꢀamino acid from TrtꢀCl to avoid the
presence of Trt esters as impurities.22 Although, this strategy renders good results, it involves an
extra step in addition to the use of organic solvents such as dichloromethane (DCM).21ꢀ22 Years
later, Suresh et al. proposed the preparation of Fmocꢀamino acids with FmocꢀCl in the presence
of activated Znꢀdust, which allows to carry out the reaction under neutral conditions.23ꢀ24
However, most commonly used method for the synthesis of Fmocꢀamino acids out of the
mentioned above protocols, is FmocꢀOSu (or NHS) which was questioned when Hlebowicz et al.
at Bachem Europe25 showed that FmocꢀAAꢀOH prepared from FmocꢀOSu contained Fmocꢀβꢀ
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AlaꢀOH and FmocꢀβꢀAlaꢀAAꢀOH formed through a Lossen rearrangement after the successive
attack of OSu on one of the carbonyls of the HOSu moiety present in FmocꢀOSu (Figure 2).
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Figure 2: Mechanism of formation of βꢀAlanine via Lossen's rearrangement
These findings revitalized again the research for developing new reagents and/or strategies for a
safe introduction of the Fmoc group. Table 1 enlists different Fmoc derivatives used for the
protection of amino acids and results of their application for the preparation of FmocꢀGlyꢀOH.
Although, this reaction could take place with all amino acids,18ꢀ19 it is more pronounced with
Gly, since its low sterical hindrance favors a high percentage of oligomerization. These Fmocꢀ
introducing derivatives should present two key characteristics: (i) no special high reactivity to
avoid the formation of oligopeptides and (ii) the leaving group, normally a hydroxyl compound,
which is displaced by the amino acid, should be easily removable during the workꢀup. Thus, our
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group, first proposed Fmocꢀ2ꢀmercaptobenzothiazole (FmocꢀMBT), which give rise to the Fmoc
derivatives with minimal contamination of oligomers26 (Table 1, #4). However, the poor
solubility of the released 2ꢀMBT byproduct required washings with organic solvents for its total
removal, which also dissolve the prepared Fmocꢀamino acid, and therefore was detrimental for
the final yield. Similar problem was encountered by Verlander, Goodman, et al where the
alcohol was contaminating the final product with low overall yields (4ꢀ30%) when
(poly)chlorophenyl derivatives were used19.
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Table 1. Fmocꢀprotecting reagents (FmocꢀOX)
Fmoc-Gly-OH
Fmoc-Gly-Gly-OH
(%)
#
Fmoc-X
[Ref.]
Scale
(Mmol)
7.6
Yield (%)
1
10ꢀ20
2ꢀ6
89
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25
NAa
NAa
78
10ꢀ20
NDb
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NAa
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20
0.55
68
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Traces
NDb
550
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55c
NDb
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NAa
NAa
NAa
NAa
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88
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NAa
NDb
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11.2
NAa
NAa
NA
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NDb
NDb
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94
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90.8
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aNA: not available; ND: no detected; to remove the 2ꢀMBT, which was not soluble in the aqueous
phase, the final product should be washed with solvent, which also dissolved the Fmocꢀamino acid.
b
c
Other succinimidyl based derivatives such as the Phtalimide,19, 29 the norbornenyl (which is the
radical derived from norbornene),30 and the corresponding spiro analogue, a six member
derivative31 were also assayed but with no remarkable advantage (Table 1, #5ꢀ7). In parallel, our
group detected an important formation of a substituted βꢀalanine residue when the norbornenyl
was used in combination with EDC for the solidꢀphase peptide synthesis (SPPS) in H2O.38 In
order to overcome the challenging problem of β–alanine residues insertion when succinimidyl
derivatives are used, Najera et al. has described a polymeric version of the reagent in which the
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βꢀalanine contamination remained anchored to the polymeric support.39ꢀ40 This bright strategy
limits its usage in the preparation of small amounts of protected amino acids.
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Other active species commonly used with carbodiimide based couplings such as
pentafluorophenyl (Pfp)19, 32 or benzotriazole (Bt)33 were also addressed, with good yields,
though the reagents are either expensive (Pfp) or explosive (Bt), respectively (Table 1, #8,9).
Even Fmocꢀtriazine derivative was investigated34 (Table 1, #10) as a method.
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For Fmoc introduction using neat aqueous solution, Fmocꢀphenyldimethylsulfonium methyl
sulfate (FmocꢀODsp)35 has been used but no formation of dipeptides was studied (Table 1, # 11).
Recently, our group described OxymaPure® as superior additive for the carbodiimide based
coupling strategy.41 This reagent competes favorably in front of the traditionally used HOBt,42
which is being discontinued due to its explosive properties.43 Inspired by the impressive
reactivity of OxymaPure® and it’s fast assimilation in the peptide industry, we investigated other
less reactive oxime derivatives for the introduction of Fmoc group.36ꢀ37 From the first screening
(Table 1, #12ꢀ16),36 which also included 2ꢀhydroxypyridine Nꢀoxide (HOPO), the OxymaPure®
derivative (Table 1, #12), as expected due to its high reactivity, gave the largest amount of
dipeptide, followed by HOPO (Table 1, #16). The best candidate was the cyanopyridyl oxime
(Table 1, #15), but this is an expensive derivative and it can be difficult to remove from the
reaction medium. The second screening gave overall less amount of dipeptide formation (Table
1, #17ꢀ20)37 and from this the cyano amide derivative (Amox) (Table 1, #20) was selected as the
candidate to substitute HOSu for the introduction of Fmoc. Amox synthesis is inexpensive and
can be easily removed after the reaction as the solubility of Amox in water was found to be 0.9
M which assures that it will not contaminate the final Fmocꢀamino acid, unlike the case of MBT
(Table 1, #4).
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Initially FmocꢀAmox was prepared following the similar methodology used for other derivatives
with slight modifications. The reaction of FmocꢀCl and Amox in presence of sodium carbonate
(aq solution) using DCM as solvent was carried out for 20 h at room temperature. After
completion of reaction (monitored by HPLC for complete consumption of FmocꢀCl), the reaction
mixture was diluted with DCM. The reaction mixture in separating funnel prolonged the
separation of DCM from aqueous phase. However, after separation the organic phase was
collected and solvent was removed under vacuum. The HPLC chromatogram clearly showed
high purity of FmocꢀAmox synthesis. FmocꢀAmox has been shown to be stable at room
temperature for several months.
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FmocꢀGlyꢀOH was prepared at two different scales using 1 and 40 g with similar results. In
terms of Fmocꢀdipeptide formation, HꢀGlyꢀOH proves to be an excellent candidate for
evaluating the performance of FmocꢀAmox, since its low sterical hindrance favors a high
percentage of oligomerization.39 The reaction procedure is described as follows: The solution of
FmocꢀAmox in acetone was added slowly to a stirring solution of HꢀGlyꢀOH and Na2CO3 in
water. The pH of the reaction mixture was maintained at 9ꢀ10 by successive addition of Na2CO3.
The reaction was monitored by TLC as well as by pH stability (decrease of pH was measured as
a sign which indicated that the reaction was taking place). After completion of the reaction (4 h),
solvent was removed and the remaining aqueous layer was washed with DCM, followed by
addition of 1N HCl (till pH <2) which afforded offꢀwhite precipitate. The precipitate was then
filtered and recrystallized using ethylacetate and nꢀHexane (93 %) (see Experimental Section for
more detailed procedure).
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Fmoc-Gly-Gly-OH
Fmoc-Gly-OH
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No formation
of Fmoc-Gly-
Gly-OH
A
B
Figure 3: A. HPLC Chromatogram of FmocꢀGlyꢀOH and coꢀeluted FmocꢀGlyꢀGlyꢀOH. B.
HPLC Chromatogram of synthetic FmocꢀGlyꢀOH.
The purity of FmocꢀGlyꢀOH was monitored by HPLC. FmocꢀGlyꢀGlyꢀOH, which was prepared
by solidꢀphase technique, was also eluted along with FmocꢀGlyꢀOH to indicate the formation of
any detrimental impurity of dipeptide. As shown in Figure 3, there was no trace of dipeptide
observed in the chromatogram (Figure 3). Futhermore, 1HꢀNMR did not showed the presence of
any contamination of Amox (Figure 4), which clearly indicates the advantage of Amox for the
use in the synthesis of Fmocꢀamino acids.
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Figure 4: Comparison of 1H and 13C NMR of FmocꢀGlyꢀOH, FmocꢀAmox and Amox.
Prompted by these results, FmocꢀPheꢀOH and FmocꢀValꢀOH (10 g scale each) were also
prepared using the procedure described above (Figure 5 and 6). The purity of the final product
was confirmed by HPLC and NMR which shows no trace formation of dipeptide. Importance of
solvent during workup was also studied as DCM is considered to be toxic and not suitable for
bulk scale up reactions. Ethyl acetate was found to be a suitable substitute for DCM. Ethyl
acetate layer when used during workup was collected, dried over MgSO4, filtered and
concentrated under vaccum. However, it was found to contain trace amount of Amox (as
observed by NMR), which is not detrimental in the peptide synthesis. Therefore, the obtained
product was dissolved in DCM and extracted using distilled water. The DCM workup helps in
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the removal of trace amount of Amox yielding pure compound as confirmed by NMR (Figure
7).
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Fmoc-Phe-Phe-OH
Fmoc-Phe-OH
No
formation
of Fmoc-
Phe-Phe-
OH
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Fmoc-Phe-OH
Fmoc-Amox
Amox
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Figure 5I: A. HPLC Chromatogram of FmocꢀPheꢀOH coꢀeluted with FmocꢀPheꢀPheꢀOH. B.
HPLC Chromatogram of FmocꢀPheꢀOH
Figure 5II. Comparison of 1H and 13C NMR of FmocꢀPheꢀOH, FmocꢀAmox and Amox
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I
Fmoc-Val-Val-OH
Fmoc-Val-OH
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Figure 6I: A. HPLC Chromatogram of FmocꢀValꢀOH coꢀeluted with FmocꢀValꢀValꢀOH and
B. HPLC Chromatogram of FmocꢀValꢀOH
Figure 6II. Comparison of 1H and 13C NMR of FmocꢀValꢀOH, FmocꢀAmox and Amox
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DCM Extraction
EtOAc Extraction
Amox
Figure 7: Comparison of workup using ethyl acetate and DCM for extraction of FmocꢀPheꢀOH
These results clearly indicate the proficiency and superiority of FmocꢀAmox in synthesizing
Fmocꢀamino acids, which are most prone to give the sideꢀreaction.
Conclusion
In order to overcome one of the biggest challenges in the synthesis of peptides building blocks,
which is the undesired formation of diꢀ and tripeptides during the protection of amino acids,
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various methods incorporating large array of FmocꢀLeaving Groups have been intensively
studied. Although few of the addressed reagents showed promising result, none of them has
turned on to be a potential candidate for the industrial application. In this work, we are proposing
FmocꢀLeaving Group derivative, FmocꢀAmox, based on the structure of wellꢀknown
carbodiimide additive family – oxime. FmocꢀAmox has been used for Fmoc protection in case of
HꢀGlyꢀOH, a challenging model which favors the formation of side reactions, showed
outstanding performance with excellent yield (93 %) and total absence of detrimental impurities
in the form of Fmocꢀdipeptide, as supported by the HPLC and NMR analysis. Furthermore, it is
envisaged that Amox derivatives will be useful for the introduction of other protecting groups
such as pNZ, Alloc, Boc.
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Experimental Procedures
Materials
The solvents used were of HPLC reagent grade and purchased from commercial suppliers. Magnetic
resonance spectra (1H, 13C, DEPT90, DEPT135) were recorded with Bruker 400 MHz, chemical shift
values reported in δ units (ppm) using TMS as internal standard. Followꢀup of the reactions and checks of
the purity of the compound was done by TLC on silicaꢀgelꢀprotected aluminum sheets 60 F254 (Merck),
and the spots were detected by exposure to UV light at λ = 254 nm. Analytical HPLC was performed on
Agilent 1100 system using Phenomenax C18 column (3µm, 4.6 × 50 mm) and for data processing
Chemstation software was used. Buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA in CH3CN were used
in HPLC. LCMS was performed on Shimadzu 2020 UFLC using YMCꢀ Triart C18 (5µm, 4.6 × 150 mm)
column and data processing was carried out by Lab Solution software. Buffer A: 0.1% formic acid in
H2O, buffer B: 0.1% formic acid in CH3CN were used.
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Synthesis
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Synthesis of Fmoc-Amox: A solution of FmocꢀCl (5 mmol) in 15 mL DCM was added slowly to a
solution of 5.5 mmol of Amox and sodium carbonate (10 mmol) in 10 mL water with stirring at 0 °C for
30 min and then at room temperature for 20 h. After dilution with DCM, the organic phase was collected
and washed with water and saturated brine solution. DCM layer was dried over MgSO4, filtered and the
solvent was removed to obtain pure FmocꢀAmox (80% yield).
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HPLC [5 ꢀ 95 % of CH3CN (0.1% TFA)/ H2O (0.1 % TFA)] retention time (tR) = 10.22 min. 1H
NMR (400 MHz, DMSO-d6) 4.45 (1H, t, J = 6.0 Hz), 4.83 (2H, d, J = 6.0 Hz), 7.36 (2H, t, J =
7.2 Hz), 7.44 (2H, t, J = 7.2 Hz), 7.70 (2H, d, J = 7.4 Hz), 7.92 (2H, t, J = 7.4 Hz), 8.24 (2H, brs
13
NH). C NMR (100 MHz, DMSO-d6) 157.4 (C), 150.5 (C), 142.7 (C), 140.7 (C), 134.5 (C),
127.9 (CH), 127.2 (CH), 124.8 (CH), 120.2 (CH), 107.5 (C), 70.8 (CH2), 46.0 (CH)
Synthesis of Fmoc-Amino acid: A solution of FmocꢀAmox (119.4 mmol in case of Gly and 30 mmol in
case of Phe and Val) in acetone was slowly added to the stirred solution of amino acid (131.34 mmol in
case of Gly and 33 mmol for Phe and Val) and sodium carbonate (106 mmol in case of Glycine and 75
mmol in case of Phe and Val) in distilled water. Constantly, pH (9ꢀ10) was monitored and reaction
mixture was maintained basic. Reaction was monitored by TLC till the complete consumption of Fmocꢀ
Amox. After 4 h stirring at room temperature, the reaction mixture was concentrated under reduced
pressure to remove acetone and then extracted with DCM to remove unreacted FmocꢀAmox. The reaction
mixture was then acidified with 1N HCl to give offꢀwhite solid, which was filtered and washed with water
several times. The filtrate was dried and recrystallized (ethyl acetate/ hexane) to afford pure Fmoc
protected amino acid. The purity of Fmocꢀamino acids were determined by HPLC analysis using a
Phenomenax C18 column (3 µm, 4.6 × 50 mm) with a linear gradient over 15 min, flow rate: 1.0 mL/min,
1
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detection at 220 nm and were characterized by H, C, DEPT90 & DEPT135 NMR (Spectra attached in
Supporting information).
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FmocꢀGlyꢀOH: HPLC [5 ꢀ 95 % of CH3CN (0.1% TFA)/ H2O (0.1 % TFA)] retention time (tR)
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= 8.69 min. H NMR (400 MHz, DMSO-d6) 3.67 (2H, d, J = 6 Hz), 4.24 (1H, t, J = 6.4 Hz),
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4.31 (2H, d, J = 7.2 Hz), 7.34 (2H, t, J = 7.2 Hz), 7.43 (2H, t, J = 7.2 Hz), 7.64 (NH, t, J = 6 Hz),
7.72 (2H, d, J = 7.2 Hz), 7.90 (2H, d, J = 7.2 Hz), 12.56 (COOH).13C NMR (100 MHz, DMSO-
d6) 171.5 (C), 156.4 (C), 143.8 (C), 140.6 (C), 127.5 (CH), 127.9 (CH), 125.1 (CH), 120.0 (CH),
65.6 (CH2), 46.5 (CH), 42.0 (CH2).
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FmocꢀPheꢀOH: HPLC [5 ꢀ 95 % of CH3CN (0.1% TFA)/ H2O (0.1 % TFA)] retention time (tR)
1
= 10.92 min. H NMR (400 MHz, DMSO-d6) 2.88 (1H, t, J = 11.4 Hz), 3.09 (1H, d, J = 13.8
Hz), 4.19 (4H, m), 7.28 (7H, m) 7.40 (2H, t, J = 7.2 Hz), 7.65 (2H, t, J = 7.4 Hz), 7.74 (NH, d, J
= 8.3 Hz), 7.88 (2H, d, J = 7.5 Hz), 12.75 (COOH). 13C NMR (100 MHz, DMSO-d6) 173.3 (C),
155.9 (C), 143.7 (C), 140.6 (C), 137.9 (C), 129.0 (CH), 128.1 (CH), 127.5 (CH), 127.0 (CH),
126.3 (CH), 125.2 (CH), 120.0 (CH), 65.5 (CH2), 55.4 (CH), 46.5 (CH), 36.4 (CH2).
FmocꢀValꢀOH: HPLC [20 ꢀ 65 % of CH3CN (0.1% TFA)/ H2O (0.1 % TFA)] retention time (tR)
= 11.99 min. 1H NMR (400 MHz, DMSO-d6) 0.90 (6H, d, J = 6.7 Hz), 2.08 (1H, m), 3.87 (1H,
m), 4.25 (3H, m), 7.32 (2H, m) 7.41 (2H, t, J = 7.4 Hz), 7.48 (NH, d, J = 8.6 Hz), 7.74 (2H, m),
7.89 (2H, d, J = 7.5 Hz). 13C NMR (100 MHz, DMSO-d6) 173.3 (C), 156.3 (C), 143.8 (C),
140.6 (C), 127.5 (CH), 127.0 (CH), 125.3 (CH), 120.0 (CH), 65.6 (CH2), 59.7 (CH), 46.6 (CH),
29.7 (CH), 19.2 (CH3), 18.0 (CH3).
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ASSOCIATED CONTENT
Electronic Supplementary Information (ESI) available: [Experimental details and HPLC
chromatograms] and these materials are available free of charge via the Internet at
http://pubs.acs.org.”
AUTHOR INFORMATION
Corresponding Author
* B. G. de la Torre. Eꢀmail: garciadelatorreb@ukzn.ac.za.
* F. Albericio. Eꢀmail: albericio@ukzn.ac.za or albericio@ub.edu.
Author Contributions
Preparation of FmocꢀAmox was done by EH, the rest of the experimental was carried out by AK
and AS. The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
ACKNOWLEDGMENT
This work was funded in part by the National Research Foundation (NRF), the University of
KwaZuluꢀNatal (South Africa), and the Generalitat de Catalunya (2014 SGR 137) and MEC
(CTQ2015ꢀ67870ꢀP). Finally, the authors thank Mr. Yoav Luxembourg (Luxembourg Bio
Technologies Ltd) for continuous support this study.
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groups. Synthesis 1987, 1987, 166ꢀ167.
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Schön, I.; Kisfaludy, L., 9ꢀFluorenylmethyl pentafluorophenyl carbonate as a useful
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