An improved method for the incorporation of fluoromethyl ketones into solid phase peptide synthesis techniques

Article abstract of DOI:10.1039/d1ra03046a

An improved and expedient technique for the synthesis of peptidyl-fluoromethyl ketones is described. The methodology is based on prior coupling of an aspartate fluoromethyl ketone to a linker and mounting it onto resin-bound methylbenzhydrylamine hydrochloride. Subsequently, by utilising standard Fmoc peptide procedures, a number of short Z-protected peptides were synthesised and assessed as possible inhibitors of the main protease from SARS-CoV-2 (3CL^pro).

Full text of DOI:10.1039/d1ra03046a

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PAPER
An improved method for the incorporation of
fluoromethyl ketones into solid phase peptide
synthesis techniques†
Cite this: RSC Adv., 2021, 11, 20457
Dhira Joshi,a Jennifer C. Milligan,b Theresa U. Zeisner,c Nicola O'Reilly,a
a
John F. X. Diffleyb and George Papageorgiou
*
An improved and expedient technique for the synthesis of peptidyl-fluoromethyl ketones is described. The
methodology is based on prior coupling of an aspartate fluoromethyl ketone to a linker and mounting it
onto resin-bound methylbenzhydrylamine hydrochloride. Subsequently, by utilising standard Fmoc
peptide procedures, a number of short Z-protected peptides were synthesised and assessed as possible
inhibitors of the main protease from SARS-CoV-2 (3CLpro).
Received 19th April 2021
Accepted 27th May 2021
DOI: 10.1039/d1ra03046a
rsc.li/rsc-advances
making it an attractive target for inhibitor design.19 Its substrate
sequence preference is known, and thus it is a good candidate
Introduction
to demonstrate the effectiveness and specicity of short PFMK
inhibitors. Since the nsp4/5 cut site is predicted to have the
highest affinity for 3CLpro, the peptidyl moiety was based on this
sequence, except for an aspartic acid instead of a glutamine at
the P1 site due to ease of synthesis.20
Although peptidyl-uoromethyl ketones (PFMKs) were postulated
to be effective serine protease inhibitors in the late 1960s,1 their
efficient and atom economic synthesis still remains a key objective
for peptide chemists. The rst syntheses of uoromethyl ketones
(FMKs) were independently reported by the Rasnick,2 Shaw3 and
Imperiali4 groups in the mid-1980s. Since then, numerous re-
ported syntheses of PFMKs have appeared in the literature
together with comprehensive reviews1,5–8 describing their wide-
spread use as probes for serine and cysteine proteases and as
potential drugs for rheumatoid arthritis9,10 and other diseases.11,12
PFMKs are well known as inhibitors of serine and cysteine
proteases, such as caspases, cathepsins and Sentrin/SUMO
specic proteases.13–15 Their peptide backbone can be altered
to mimic a substrate sequence that binds directly to the active
site of a protease. This makes PFMKs excellent tools for target-
PFMKs are usually obtained by incorporating the corre-
sponding uoroalcohol into a peptide sequence, by a solution
coupling with the carboxylic acid terminus of a peptide, fol-
lowed by alcohol oxidation.4,21–25 However, this methodology is
limited to peptide sequences that either do not contain cross-
reacting sidechain groups, have their sidechains orthogonally
protected, or do not contain oxidation sensitive residues such as
Cys, Met, Trp or Tyr. Furthermore, the solubility issue associ-
ated with fully protected amino acids in solid phase peptide
synthesis (SPPS) and the racemisation possibilities at the C-
terminal amino acid limit the capacity of this synthetic proce-
dure. Therefore, there is scope for prior formation of the amino
acid FMK moieties which can be incorporated to a peptide
sequence either by solution chemistry or by SPPS techniques.
based inhibition of specic proteases. The 3C-like protease
(3CLpro
)
of severe acute respiratory syndrome coronavirus 2
16
(SARS-CoV-2), the causative agent of the global COVID-19
pandemic, is a particularly interesting therapeutic target. The
SARS-CoV2 genome encodes two overlapping polyproteins,
from which the functional polypeptides have to be released
Results and discussion
principally by proteolytic processing by 3CLpro
.
Therefore,
17,18
this cysteine protease is essential for the viral replicative cycle.
Human proteases with similar cleavage specicity are very rare,
The most commonly reported methodology for the synthesis of
N-protected amino acid FMKs is based on the conversion of
halomethyl ketones (mostly bromo or chloro) to the corre-
sponding uoro analogues. Halomethyl ketones are rst
prepared by reacting the mixed anhydride or otherwise activated
acid with diazomethane, to give the corresponding diazoketone
which upon treatment with anhydrous HCl or aqueous HBr
affords the required halomethyl ketone. Reeder and Anderson's26
comprehensive review listed the most common reagents and
conditions for these transformations. Alternative procedures,
which avoid the use of diazomethane, also require hazardous
aPeptide Chemistry STP, The Francis Crick Institute, 1 Midland Road, London NW1
1AT, UK. E-mail: George.Papageorgiou@crick.ac.uk; Tel: +44 (0)203 796 2359
bChromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road,
London NW1 1AT, UK
cCell Cycle Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT,
UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ra03046a
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reagents and conditions. Diazomethane still remains the most
Aspartic acid uoromethyl ketone (D-FMK) is an important
effective reagent for this transformation despite associated safety synthon for the construction of highly desirable short peptides
concerns due to its toxicity, thermal instability and shock sensi- such as Z-VAD-(OMe)-FMK, which is used as a caspase inhib-
tivity, especially in large scale reactions. TMS–CHN2 is consider- itor.29,30 Our initial strategy was to prepare compound 1 in
ably less toxic than diazomethane itself, but it has been a useful quantity and use it as the rst amino acid unit in
reported27 that it does not react with mixed anhydrides derived standard Fmoc-SPPS protocols. The aim was to attach it to the
from amino acids, possibly due to steric hindrance.
resin, construct the required peptide and methylate the acid
As a part of our aim to synthesise a number of short Z-protected side group aer cleavage from the solid support. As depicted in
peptidyl-FMKs we initially based our investigations on a published Scheme 1, starting from the commercially available Fmoc-Asp-
procedure28 which claimed the synthesis of uoromethyl dime- (OtBu)-OH we prepared the bromomethyl ketone 2 in multigram
thylketal 1. We envisioned to use this reagent in SPPS procedures quantity in an excellent yield in a tandem reaction manner
to construct the desired short peptides. We present here the without isolating the intermediate diazoketone derivative.
shortcomings encountered with this approach and report an Although the conversion of 2 to the uoromethyl ketone 3, was
alternative new viable methodology for the synthesis of PFMKs.
reported28 at 49% yield, in our hands gave variable 18–38%
yields. A brief effort to improve the yield of this reaction using
a variety of uoride sources such as KF, LiF, or TBAF under
various conditions (data not shown) resulted either in complete
cleavage of the Fmoc group or no reaction. With uoromethyl
ketone 3 in hand we proceeded to protect the ketone func-
tionality as a dimethylketal, as described28 with the objective of
obtaining compound 1. To our initial surprise the only isolated
product proved to be the lactone 4. The structure of the product
Scheme 1 Synthesis of key synthon 6. Reagents and conditions: (i) iso-butyl chloroformate, NMM, THF, ꢀ10 ꢁC; (ii) CH2N2, Et2O, 0 ꢁC to rt; (iii)
33% HBr in AcOH–H2O (1 : 2); (iv) TBAF, p-TsOH, THF, reflux; (v) p-TsOH, MeOH, toluene, 60 ꢁC; (vi) CH2Cl2, TFA, 0 ꢁC; (vii) Me3SiCl, MeOH; (viii)
2,2-dimethoxypropane, MeOH, p-TsOH, 60 ꢁC.
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Scheme 2 Proposed mechanism for the formation of 4. Reagents and conditions: 10% p-TsOH, MeOH, 60 ꢁC.
was conrmed by both 1-dimensional high eld 1H, 13C and 19
F
The failure to prepare the desired FMK 1 prompted us to seek
NMR spectroscopy and also by 1H–1H COSY and 1H–13C HSQC, an alternative approach. Since the protection of the ketone
correlation spectroscopy (see ESI†). Halomethyl ketones 3 and 2 functionality as a ketal proved problematic, we decided to use
were easily transformed to the corresponding methyl esters 6 Fmoc-Asp(OMe)-FMK as our key synthon, which enabled
and 8 respectively, either by isolating the corresponding acids 5 shorter synthesis and atom economy from commercially avail-
and 7, or by in situ deprotection with TFA and methylation with able Fmoc-Asp(OMe)-OH. Thus, bromomethyl ketone 8 was
TMS–Cl in MeOH. The use of these reagents in this study is obtained in an excellent yield using the same one-pot procedure
discussed below.
described above. Although conversion to FMK 6 was accom-
In order to further investigate this unexpected cyclisation, we plished in relatively low yield, owing to the issues described
attempted ketone protection on the more readily available above, the shorter synthetic pathway compensates for this
bromomethyl ketone 2 under a variety of appropriate condi- shortcoming (Scheme 3).
tions, using either methanol or 2,2-dimethoxypropane for ketal
It was then envisaged that the best way forward was to
formation. In all cases, the isolated product was the lactone 9. reversibly anchor the ketone to an easily accessible linker which
We also found that attempting to form a cyclic ketal using could be attached to an appropriately functionalised resin. The
ethylene glycol predominantly gave a lactone (data not shown). 1,3-diol linker described by Funeriu et al.28 was not an attractive
We therefore concluded that the intermediate dimethyl ketal 3a option because of the extensive synthetic work required, and the
(Scheme 2) generated from uoromethyl ketone 3, is unstable harsh conditions (37% HCl) needed for cleavage. Quibell and
under the mild acidic conditions. Cyclisation with concomitant co-workers31 reported the condensation of bicyclic ketones with
loss of methanol and isobutylene generates the thermody- the hydrazide linker 10 described by Murphy et al.32 This was
namically stable lactone 4. Our proposed mechanism is depic- considered more suitable due to its robustness and straight-
ted in Scheme 2. Despite using similar experimental conditions forward short synthesis. Also, the ease of cleavage using rela-
Funeriu et al.,28 did not observe the formation of 4 and reported tively mild conditions made this method more attractive. Thus,
the isolation of 1 in 77% yield. Nonetheless, they did not report the linker 10 was prepared in multigram quantity from
its incorporation into peptide sequence either by solution commercially available tranexamic acid in an excellent overall
chemistry or by SPPS methods. In fact, they protected the yield (see ESI†). Condensation of FMK 6 with 10 gave the desired
ketone function of Fmoc-glycidyl-FMK as a cyclic acetal which hydrazone 11 in good yield. To conrm the viability of this
was then attached to a linker via a click chemistry reaction.
approach,
11
was
coupled
to
resin-bound
4-
Scheme 3 Shorter synthesis of key synthon 6. Reagents and conditions: (i) iso-butyl chloroformate, NEM, THF, ꢀ10 ꢁC; (ii) CH2N2, Et2O, 0 ꢁC to
rt; (iii) 33% HBr in AcOH–H2O (1 : 2); (iv) TBAF, p-TsOH, THF, reflux.
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Scheme 4 Synthesis of FMK-peptides 13–16. Reagents and conditions: (i) NaOAc, EtOH, H2O, reflux; (ii) HATU, DMF, DIPEA; (iii) non-automated
SPPS using Fmoc aa, HATU, DIPEA, DMF; (iv) 95% TFA, 2.5% H2O, 2.5% TIPS; (v) fully automated SPPS using Fmoc aa, HATU, DIPEA, DMF.
methylbenzhydrylamine (MBHA) hydrochloride to form 12. We concentration of the PFMK inhibitors 3CLpro protease activity is
rst prepared the commercially available Z-VAD(OMe)-FMK 13 completely abolished, even at the lowest concentration tested
using non-automated SPPS protocols. Subsequently with the (25 mM). This demonstrates that PFMKs might be a useful tool
effectiveness of the method established, we used fully auto- for probing 3CLpro enzymatic activity. However, their develop-
mated SPPS protocols to synthesise a further three short ment into clinical drugs is unlikely, due to their high in vivo
peptides, Z-AVLD(OMe)-FMK 14, Z-SAVLD(OMe)-FMK 15 and Z- toxicity, which might be explained by the metabolic conversion
ASAVLD(OMe)-FMK 16. The purity of all peptides was estimated of the FMK group into toxic uoroacetate.34
by integration of the peak from LCMS analysis (Scheme 4).
The usefulness of the synthesised short peptides 13–16 as
Conclusions
inhibitors of 3CLpro of SARS-CoV-2 was investigated and full
disclosure of biological results will appear elsewhere.33 An
example of the use of Z-SAVLD(OMe)-FMK 15 against the SARS-
CoV-2 3CLpro main protease is disclosed here. The in vitro
inhibitory effect of the 3CLpro specic PFMK was investigated
using a gel-based protease assay. Briey, a fusion protein was
constructed from non-structural protein 9 (nsp9) of SARS-CoV-2
with an N-terminal FLAG-His epitope. A short linker sequence
based on the natural nsp4/5 cleavage site connects the epitope
and nsp9. Upon incubation with 3CLpro, the FLAG-His-tag is
cleaved off from nsp9 at this cleavage site, resulting in two
products visible at approximately 11 kDa and 16 kDa in the
absence of the inhibitor (Fig. 1). In the presence of varying
In this paper we describe an improved general and efficient
method for the preparation of PFMKs using standard Fmoc
SPPS chemistry. This methodology permits the efficient intro-
duction of aspartate uoromethyl ketone at C-terminal end of
any peptide sequence. It also demonstrates that conversion of
the a-carboxyl group to uoromethyl ketone functionality is
feasible to most Fmoc protected amino acids prior to appro-
priately side chain group protection where is a prerequisite. The
ease of condensation of the ketone function with the func-
tionalised hydrazine linker allows the introduction of any
amino acid as rst unit in the sequence. Subsequent coupling to
resin-bound MBHA followed by standard Fmoc-SPPS tech-
niques will give access to the desired PFMK with any amino acid
at the C-terminal. The usefulness of the technique is demon-
strated by the synthesis of short peptides 13–16 and their
application to current investigations for a therapeutic target of
SARS-CoV-2.
Experimental section
General experimental information
Air or water sensitive reactions were performed in anhydrous
solvents under a positive pressure of nitrogen. Dried and other
solvents, reagents and resins were purchased from commercial
Fig. 1 Gel based cleavage assay.
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suppliers and used without further purication. NMR spectra
C
24H26NBrO5: C, 59.02; H, 5.37; N, 2.37. Found: C, 59.57; H,
were recorded on Bruker Avance III spectrometers operating at 5.24; N, 2.99%.
400, 600, 800 and 950 MHz in CDCl3 solution with TMS as
tert-Butyl (S)-3-{[(9H-uoren-9-yl)methoxycarbonyl]amino}-
internal reference, unless otherwise specied. Chemical shis 5-uoro-4-oxopentanoate 3. To a solution of 2 (3.91 g, 8
(d) are reported in parts per million (ppm). Multiplicities are mmol) and p-TsOH$H2O (4.59 g, 24 mmol) in THF (120 mL) was
abbreviated as broad (br) singlet (s) doublet (d) triplet (t) added dropwise a solution of TBAF$H2O (12.55 g, 48 mmol) in
quadruplet (q) and multiplet (m). Elemental analyses were THF (120 mL) and the mixture was stirred in a nitrogen atmo-
carried out by MEDAC Ltd., Surrey, UK. Thin layer chromatog- sphere under reux overnight. Aer cooling to rt the solvent was
raphy (TLC) was performed on Merck silica gel coated glass F254 evaporated, and the residue was dissolved in EtOAc (200 mL)
plates and were visualized by UV irradiation at 254 nm. and washed with water and brine. The organic phase was dried
Preparative column chromatography was performed on Biotage and evaporated, and the residue was ash chromatographed
Isolera One 3.3.0. All extracts of organic solvents were dried over [EtOAc–hexanes (1 : 9) to (1 : 4)] to give 3 (ref. 28) (1.29 g, 38%)
anhydrous Na2SO4 and evaporated under reduced pressure.
Diazomethane generation should be carried out in a fast-
as pale crystals, mp 56–58 ꢁC (Et2O–hexanes). Anal. Calcd for
24H26NFO5: C, 67.43; H, 6.13; N, 3.28. Found: C, 67.42; H,
C
ow ventilated fume cupboard using only glassware with 6.09; N, 3.41%.
Clear-Seal joints (no grinded joints) and without any means of
(9H-Fluoren-9-yl)methyl [(3S)-2-(uoromethyl)-2-methoxy-5-
stirring. All necessary safety precautions should be strictly oxotetrahydrofuran-3-yl]-carbamate 4. To a solution 3 (0.92 g,
applied! The generated amount of reagent should be used 2.15 mmol) in toluene (92 mL) and MeOH (9.2 mL) was added p-
immediately avoiding any long-term storage.
TsOH$H2O (41 mg, 0.215 mmol) and the mixture was heated
For diazomethane generation and use in the experiment under reux connected to a Dean–Stark trap overnight. Aer
described below, a mixture of 2-(2-ethoxyethoxy) ethanol (84 16 h more MeOH (3 ꢂ 15 mL) and p-TsOH$H2O (2 ꢂ 50 mg)
mL) and ether (48 mL) was added to a solution of KOH (15 g) in were added and reuxing continued for 48 h. Aer cooling to rt
water (24 mL) in a 500 mL round bottom ask attached to the solvent was concentrated to about half its original volume
a condenser and receiving ask without stirring or edging. The and washed with 5% aq. NaOAc and brine. The organic phase
ꢁ
reaction vessel was warmed to 65 C on water-bath and a solu- was dried and evaporated to a light brown oil (0.96 g). Flash
tion of Diazald (N-methyl-N-nitroso-p-toluenesulfonamide, 30 g) chromatography [EtOAc–hexanes (1 : 4) to (2 : 3)] gave two
in diethyl ether (270 mL) was added slowly from a dropping fractions. The rst eluted material was unreacted starting
funnel in similar rate to distillation. Aer the addition of all material 2 (113 mg, 12%). The second eluted material was the
Diazald solution, more diethyl ether (60 mL) was added to the lactone 4 (504 mg, 61%) as a viscous colourless oil which crys-
dropping funnel and distillation continued until the distillate tallised to white solid upon trituration with Et2O–hexanes, mp
became colourless. The distilled ethereal yellow solution of 110–111 ꢁC (Et2O–hexanes). Anal. Calcd for C21H20NFO5: C,
1
diazomethane was collected in a 500 mL conical ask which was 65.45; H, 5.23; N, 3.63. Found: C, 65.39; H, 5.05; N, 3.6%. H
pre-cooled to ꢀ70 ꢁC in an acetone-dry ice bath containing KOH NMR (800 MHz): d 7.78 (d, J ¼ 7.5 Hz, 2H, HAr), 7.59 (t, J ¼
pellets. The generated diazomethane solution was then trans- 6.2 Hz, 2H, HAr), 7.42 (t, J ¼ 7.4 Hz, 2H, HAr), 7.34 (t, J ¼ 7.4 Hz,
ferred via a cannula under nitrogen to the reaction vessel and 2H, HAr), 5.75 (d, J ¼ 8.0 Hz, 1H, NH), 5.11 (dd, JH–F ¼ 47.0 Hz,
used immediately.
JH–H ¼ 16.4 Hz, CHHF), 5.00 (dd, JH–F ¼ 47.0 Hz, JH–H ¼ 16.4 Hz,
tert-Butyl (S)-3-{[(9H-uoren-9-yl)methoxycarbonyl]amino}- CHHF), 4.66–4.69 (m, 1H, CH–NH), 4.56 [dd, J ¼ 10.6, 6.7 Hz,
5-bromo-4-oxopentanoate 2. To
a
solution of Fmoc-L- 1H, CHH(Fmoc)], 4.46 [dd, J ¼ 10.6, 6.7 Hz, 1H, CHH(Fmoc)],
Asp(OtBu)-OH (12.34 g, 30 mmol) in dry THF (150 mL), cooled to 4.22 [t, J ¼ 6.5 Hz, 1H, CH(Fmoc)], 3.70 (s, 3H, OCH3), 3.08 (dd, J
ꢀ10 ꢁC was added N-methylmorpholine (NMM) (3.79 g, 37.5 ¼ 17.3, 4.3 Hz, 1H, 4-H), 2.87 (dd, J ¼ 17.3, 4.3 Hz, 1H, 4-H). 13
C
mmol) and iso-butyl chloroformate (4.71 g, 34.5 mmol). A white NMR (201 MHz): d 202.67 (d, JC–F ¼ 17.1 Hz, 2-C), 171.58 (5-C),
precipitate was formed instantly, and the mixture was stirred at 155.87 [C]O(Fmoc)], 143.59 and 143.50 (CAr), 141.41 and
ꢀ10 ꢁC for 1 h. Diazomethane (extra caution should be applied 141.39 (CAr), 127.87 and 127.85 (CHAr), 127.13 and 127.11
for the generation and handling of this reagent!), generated in (CHAr), 124.99 and 124.89 (CHAr), 120.09 and 120.08 (CHAr),
situ using the procedure described in Aldrich Technical Bulletin 84.13 (d, JC–F ¼ 183.7 Hz, CH2F), 67.09 [CH2(Fmoc)], 54.05 (3-C),
(AL-180), (99.6–128.4 mmol), in Et2O dried over KOH pellets, 52.30 (OCH3), 47.25 [CH(Fmoc)], 35.33 (C-4). 19F NMR (376
was added slowly to the stirred reaction mixture at 0 ꢁC. The MHz): d ꢀ231.79 [t (overlapped dd), JH–F ¼ 47.7 Hz]. The
solution was allowed to warm to rt overnight and then cooled to structure of the product was also conrmed by 1H–1H COSY and
0 ꢁC and treated with a solution of 33% HBr in AcOH and water 1H–13C HSQC spectra (see ESI†).
(1 : 2) (90 mL). The reaction mixture was rst stirred at 0 ꢁC for
(S)-3-{[(9H-Fluoren-9-yl)methoxycarbonyl]amino}-5-uoro-4-
1 h and then at rt for further 1 h until the evolution of gas oxopentanoic acid 5. To a solution of 3 (748 mg, 1.75 mmol) in
ceased. The mixture was diluted with EtOAc (300 mL) and CH2Cl2 (50 mL), cooled to 0 ꢁC was added TFA (10 mL) and the
washed sequentially with water, saturated aq. NaHCO3 and mixture was stirred at 0 ꢁC for 0.5 h. The solution was then
brine. The organic phase was dried and evaporated to a pale concentrated, and the residue was ash chromatographed
viscous oil which crystallised aer trituration with ether. [EtOAc–hexanes (1 : 4) to (3 : 2)] to give 5 (581 mg, 89%) as white
Recrystallization afforded 2 (ref. 28) (13.61 g, 93%) as white foam-solid. 1H NMR (400 MHz): d 7.77 (d, J ¼ 7.3 Hz, 2H, HAr),
crystals, mp 114–116 ꢁC (EtOAc–hexanes). Anal. Calcd for 7.59 (d, J ¼ 7.3 Hz, 2H, HAr), 7.39 (t, J ¼ 7.4 Hz, 2H, HAr), 7.34 (dt,
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J ¼ 7.8, 0.9 Hz, 2H, HAr), 5.33 (br s, 1H, NH), 4.32–4.70 [m, 5H, d 199.8 (Cq, C]O), 171.8 (Cq, C]O), 155.9 (Cq, C]O), 143.5 (Cq,
CH2F, CH–NH, CH2(Fmoc)], 4.19 [t, J ¼ 6.4 Hz, CH(Fmoc)], 2.85– CAr), 141.4 (Cq, CAr), 127.9 (CH, CAr), 127.1 (CH, CAr), 124.9 (CH,
3.12 (m, 1H, half CH2CO2H), 2.58–2.77 (m, 1H, half CH2CO2H). CAr), 120.1 (CH, CAr), 67.0 (CH2Br), 54.5 (CH) 52.3 (CH3), 47.3
19F NMR (376 MHz): d ꢀ231.85 [t (overlapped dd), JH–F ¼ 47.7 (CH), 35.5 (CH2), 32.3 (CH2). Anal. Calcd for C21H20NBrO5: C,
Hz].
Methyl (S)-3-{[(9H-uoren-9-yl)methoxycarbonyl]amino}-5-
56.52; H, 4.52; N, 3.14. Found: C, 56.64; H, 4.59; N, 3.16.
(9H-Fluoren-9-yl)methyl [(3S)-2-(bromomethyl)-2-methoxy-5-
uoro-4-oxopentanoate 6. To a solution of 3 (855 mg, 2 mmol) oxotetrahydrofuran-3-yl]-carbamate 9. To
a solution of 2
in CH2Cl2 (20 mL), cooled to 0 ꢁC was added TFA (5 mL) and the (0.73 g, 1.5 mmol) in MeOH (20 mL) and 2,2-dimethoxypropane
mixture was stirred at 0 ꢁC for 0.5 h. The solution was then (20 mL) was added p-TsOH$H2O (291 mg, 0.15 mmol) and the
concentrated, and the residue was re-evaporated from chloro- mixture was heated to 60 ꢁC under reux overnight. Aer
form and dried under high vacuum. The residue was then dis- cooling to rt the solvent was evaporated, and the residue diluted
solved in MeOH (25 mL) and treated with Me3SiCl (334 mg, 4 with EtOAc (50 mL) and washed with 5% aq. NaOAc and brine.
mmol) and stirred at rt overnight. The solution was concen- The organic phase was dried and evaporated to a light brown oil
trated, and the residue was ash chromatographed [EtOAc– (0.86 g). Flash chromatography [EtOAc–hexanes (1 : 4) to (1 : 3)]
hexanes (3 : 7)] to give 6 (456 mg, 59%) as a viscous colourless gave two fractions. The rst eluted material was unreacted
oil which crystallised to a white solid upon trituration with starting material 2 (391 mg, 53%). The second eluted material
Et2O–hexanes, mp 106–107 ꢁC (EtOAc–hexanes). Anal. Calcd for was the cyclic product 9 (123 mg, 18%) as white crystals, mp
1
ꢁ
C
21H20NFO5: C, 65.45; H, 5.23; N, 3.63. Found: C, 65.47; H, 140–142 C (Et2O–hexanes). H NMR (400 MHz): d 7.76 (d, J ¼
1
5.34; N, 3.72%. H NMR (600 MHz): d 7.80 (d, J ¼ 7.6 Hz, 2H, 7.5 Hz, 2H, HAr), 7.58 (dd, J ¼ 7.6, 2.8 Hz, 2H, HAr), 7.41 (t, J ¼
HAr), 7.55–7.63 (m, 2H, HAr), 7.44 (t, J ¼ 7.4 Hz, 2H, HAr), 7.35 (t, J 7.5 Hz, 2H, HAr), 7.32 (t, J ¼ 7.4 Hz, 2H, HAr), 5.82 (d, J ¼ 8.8 Hz,
¼ 7.4 Hz, 2H, HAr), 5.74 (d, J ¼ 8.2 Hz, 1H, NH), 5.13 (dd, JH–F
47.3 Hz, JH–H ¼ 16.4 Hz, CHHF), 5.01 (dd, JH–F ¼ 47.0 Hz, JH–H
¼
¼
1H, NH), 4.72 (dt, J ¼ 9.2, 4.9 Hz, 1H, CH–NH), 4.58 [dd, J ¼ 10.8,
6.5 Hz, 1H, CHH(Fmoc)], 4.46 [dd, J ¼ 10.8, 6.5 Hz, 1H,
16.4 Hz, CHHF), 4.67–4.72 (m, 1H, CH–NH), 4.58 [dd, J ¼ 10.6, CHH(Fmoc)], 4.20 [t, J ¼ 6.3 Hz, 1H, CH(Fmoc)], 4.05 (s, 2H,
6.6 Hz, 1H, CHH(Fmoc)], 4.47 [dd, J ¼ 10.6, 6.7 Hz, 1H, CH2Br), 3.68 (s, 3H, OCH3), 3.02 (dd, J ¼ 17.4, 5.0 Hz, 1H, 4-H),
CHH(Fmoc)], 4.24 [t, J ¼ 6.5 Hz, CH(Fmoc)], 3.72 (s, 3H, OCH3), 2.81 (dd, J ¼ 17.4, 4.9 Hz, 1H, 4H). 13C NMR (101 MHz): d 199.6
3.11 (dd, J ¼ 17.3, 4.3 Hz, 1H, CHHCO2Me), 2.88 (dd, J ¼ 17.2, (Cq, C]O), 171.8 (Cq, C]O), 155.9, (Cq), 143.5 (Cq, CAr), 141.4
4.3 Hz, 1H, CHHCO2Me). 19F NMR (376 MHz): d ꢀ232.00 and (Cq, CAr), 127.9 (CH, CAr), 127.1 (CH, CAr), 124.9 (CH, CAr), 120.1
231.74 [t, JH–F ¼ 47.1 Hz, Hz, rotamers (9 : 1)].
(CH, CAr), 67.0 (CH2, CH2Br), 54.5 (CH), 52.3 (CH3), 47.3 (CH),
35.5 (CH2), 32.3 (CH2).
(S)-3-{[(9H-Fluoren-9-yl)methoxycarbonyl]amino}-5-bromo-
4-oxopentanoic acid 7. To a solution 2 (635 mg, 1.3 mmol) in
Methyl (S)-3-{[(9H-uoren-9-yl)methoxycarbonyl]amino}-5-
CH2Cl2 (40 mL), cooled to 0 ꢁC was added TFA (10 mL) and the bromo-4-oxopentanoate 8. To a solution of Fmoc-L-Asp(OMe)-
mixture was stirred at 0 ꢁC for 0.5 h. The solution was then OH (7.39 g, 20 mmol) in dry THF (100 mL), cooled to ꢀ10 C
ꢁ
concentrated, and the residue was ash chromatographed was added N-ethylmorpholine (NEM) (2.88 g, 25 mmol) and
[EtOAc–hexanes (1 : 4) to (4 : 1)] to give 7 (534 mg, 95%) as pale isobutyl chloroformate (3.14 g, 25 mmol). A white precipitate
solid. 1H NMR (400 MHz): d (t, J ¼ 7.3 Hz, 2H, HAr), 7.31 (t, J ¼ was formed instantly, and the mixture was stirred at ꢀ10 ꢁC for
7.3, Hz, 2H, HAr), 5.74 [br s, 1H, NH], 4.50–4.80 [m, 5H, CH2F, 1 h. Diazomethane, generated in situ using the procedure
CH–NH, CH2(Fmoc)], 4.419 [t, J ¼ 6.4 Hz, CH(Fmoc)], 2.85–3.10 described in Aldrich Technical Bulletin (AL-180) (66.4–85.6
(m, 1H, half CH2CO2H), 2.55–3.10 (m, 1H, half CH2CO2H).
mmol) in Et2O, dried over KOH pellets, was added slowly to the
Methyl (S)-3-{[(9H-uoren-9-yl)methoxycarbonyl]amino}-5- reaction mixture at 0 ꢁC and the mixture was allowed to warmꢁto
bromo-4-oxopentanoate 8. To a solution of 2 (1.46 g, 3 mmol) rt overnight with stirring. The mixture was then cooled to 0 C
in CH2Cl2 (30 mL), cooled to 0 ꢁC was added TFA (7.55 mL) and and treated with a solution of 33% HBr in AcOH and water
ꢁ
the mixture was stirred at 0 C for 1 h. The solution was then (1 : 2) (60 mL) and stirred at 0 ꢁC for 1 h and the at rt for further
concentrated, and the residue was re-evaporated from chloro- 1 h until the evolution of gas ceased. The mixture was diluted
form and dried under high vacuum. The residue was then dis- with EtOAc (200 mL) and washed sequentially with water, brine
solved in MeOH (30 mL) and treated with Me3SiCl (0.65 g, 6 and saturated aq. NaHCO3. The organic phase was dried and
mmol) and stirred at rt overnight. The solution was concen- evaporated to a pale viscous oil which crystallised aer tritura-
trated, and the residue was ash chromatographed [EtOAc– tion with ether. Recrystallization afforded 8 (7.83 g, 88%) as
hexanes (3 : 7)] to give 8 (1.04 g, 78%) as a viscous colourless oil white crystals, mp 138–140 ꢁC (EtOAc–hexanes) identical to the
which crystallised to white solid upon trituration with Et2O– compound described above.
hexanes, mp 138–140 ꢁC (EtOAc–hexanes). 1H NMR (950 MHz):
Methyl (S)-3-{[(9H-uoren-9-yl)methoxycarbonyl]amino}-5-
d 7.79 (d, J ¼ 7.5 Hz, 2H, HAr), 7.60 (t, J ¼ 6.7 Hz, 2H, HAr), 7.43 (t, uoro-4-oxopentanoate 6. To a solution of 8 (2.23 g, 5 mmol)
J ¼ 7.3 Hz, 2H, HAr), 7.34 (t, J ¼ 7.3 Hz, 2H, HAr), 5.78 [d, J ¼ and p-TsOH$H2O (2.87 g, 15 mmol) in THF (750 mL) was added
8.5 Hz, 1H, NH], 4.74 (dt, J ¼ 4.9, 4.3 Hz, 1H, CH–NH), 4.61 [dd, J dropwise a solution of TBAF.H2O (7.84 g, 48 mmol) in THF (75
¼ 10.8, 6.6 Hz, 1H, CHH(Fmoc)], 4.48 [dd, J ¼ 10.8, 6.6 Hz, 1H, mL) and the mixture was stirred in a nitrogen atmosphere
CHH(Fmoc)], 4.23 [t, J ¼ 6.5 Hz, CH(Fmoc)], 4.07 (s, 2H, CH2Br), under reux overnight. Aer cooling to rt the solvent was
3.71 (s, 3H, OCH3), 3.05 (dd, J ¼ 17.4, 4.8 Hz, 1H, CHHCO2Me), evaporated, and the residue was dissolved in EtOAc (150 mL)
2.83 (dd, J ¼ 17.4, 4.8 Hz, 1H, CHHCO2Me). 13C NMR (101 MHz): and washed with water and brine. The organic phase was dried
20462 | RSC Adv., 2021, 11, 20457–20464
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and evaporated, and the residue was ash chromatographed Fmoc-amino acids, Z-amino acids, (5 equiv.), HATU (5 equiv.)
[EtOAc–hexanes (1 : 4) to (3 : 7)] to give 6 (0.43 g, 22%) as pale and DIPEA (10 equiv.) with respect to resin 12. The peptides
crystals, mp 106–107 ꢁC (EtOAc–hexanes) identical to the were cleaved from the resin by treatment with of an ice-cold
compound described above.
(1s,4r)-4-[(S)-11-(9H-Fluoren-9-yl)-6-(uoromethyl)-7-(2-
methoxy-2-oxoethyl)-3,9-dioxo-10-oxa-2,4,5,8-tetraazaundec-5-
cleavage solution (95% TFA, 2.5% H2O, 2.5% TIPS) for 30 min
at 0 ꢁC. Following ltration to remove the linker retaining-resin,
ether was added to the ltrate. The peptides were ether soluble
en-1-yl]cyclohexane-1-carboxylic acid 11. To a solution of semi- so following evaporation of ether the individual peptide was
carbazidyl-trans-4-methylcyclohexane carboxylic acid tri- solubilised in water and acetonitrile prior to lyophilisation.
uoroacetate salt 10 (342 mg, 1.04 mmol) in EtOH (20 mL) was Each peptide was solubilised in MeOH (1 mL) and puried on
added water (3 mL) and sodium acetate trihydrate (212 mg, 1.56 a C8 reverse phase HPLC column (Agilent PrepHT Zorbax
mmol). Fluoromethyl ketone 6 (401 mg, 1.04 mmol) was added 300SB-C8, 21.2 ꢂ 250 mm, 7 m) using a linear solvent gradient
and the mixture was heated under reux in a nitrogen atmo- of 15–65% (MeCN + 0.08% TFA) and (H2O + 0.08% TFA) over
sphere for 3 h. Aer cooling to room temperature, the solution 40 min at a ow rate of 8 mL minꢀ1. Fractions containing the
was diluted with EtOAc (80 mL) and washed with 0.1 M aq. HCl product were pooled and lyophilised to dryness and analysed by
and brine, dried and evaporated a pale foam (587 mg, 97%) LCMS on an Agilent 1100 LC-MSD (see ESI†).
which was used in the next step without further purication. 1H
NMR (400 MHz, MeOD): d 7.77–7.83 (m, 2H), 7.62–7.67 (m, 2H), MS (ES+) m/z: 581.3 [M + H]+, 603.3 [M + Na]+; calcd for
Z-AVLD(OMe)-FMK 14 (1.4 mg, 2.4%); estimated purity 37%;
7.40 (dtt, J ¼ 7.7, 3.2, 1.3 Hz, 2H), 7.28–7.36 (m, 2H), 4.97–5.19
C
28H41N4FO8: 580.3;
Z-SAVLD(OMe)-FMK 15 (0.8 mg, 1.2%); estimated purity
(m, 1H), 4.18–4.6262 (m, 4H), 3.76 (s, 1H), 3.61–3.70 (m, 2H),
2.96–3.19 (m, 1H), 2.63–3.01 (m, 2H), 2.36–2.47 (m, 1H), 2.13– 65%; MS (ES+) m/z: 668.3 [M + H]+, 690.3 [M + Na]+; calcd for
2.30 (m, 1H), 1.92–2.08 (m, 2H), 1.84 (d, J ¼ 15.4 Hz, 2H), 1.56–
1.28 (m, 2H), 1.19–1.28 (m, 3H), 0.87–1.12 (m, 2H). MS (ES+) m/z:
582.0 [M + H]+; calcd for C30H35N4FO7: 582.2.
C
31H46N5FO10: 667.3;
Z-ASAVLD(OMe)-FMK 16 (0.6 mg, 0.8%); estimated purity
33%; MS (ES+) m/z: 739.4 [M + H]+, 761.5 [M + Na]+; calcd for
Preparation of resin 12. Compound 11 (436 mg, 0.748 mmol
1.1 equiv.) was attached to resin-bound methylbenzhydrylamine
C
34H51N6FO11: 738.4.
3CLpro gel-based protease assay. Reactions were carried out
(MBHA) hydrochloride (200–400 mesh, 0.88 mmol gꢀ1 loading as described previously.33 Briey, the proteolytic activity of
capacity) (773 mg, 0.68 mmol) in DMF (3 mL) using HATU 3CLpro was assessed by monitoring the cleavage of a fusion
(272 mg, 0.714 mmol, 1.05 equiv.) and DIPEA (1.185 mL, protein substrate (FLAG-His-SAVLQ-nsp9) over time. In this
6.8 mmol, 10 equiv.) at rt for 16 h. The resin was washed with instance, 1 mM of 3CLpro in a reaction buffer containing 50 mM
DMF and used in further steps.
HEPES–KOH pH 7.6, 1 mM EDTA, 2 mM DTT, 10% glycerol, and
Synthesis of Z-VAD(OMe)-FMK 13. This peptide was syn- 0.02% Tween-20 was incubated with either varying concentra-
thesised manually by coupling resin 12 (0.14 mmol) scale with tions of Z-SAVLD(OMe)-FMK or DMSO. The cleavage reaction
Fmoc-Ala-OH followed by Z-Val-OH using standard solid phase was then initiated by the addition of 6.25 mM of FLAG-His-
Fmoc procedures. Prior to each coupling the Fmoc was removed SAVLQ-nsp9 for 1 hour at room temperature (19–23 ꢁC). The
by treating the resin with (3 ꢂ 10 mL) of 20% piperidine in DMF reactions were then quenched and denatured by the addition of
for 10 min. HATU (4.5 equiv.) and DIPEA (10 equiv.) were used SDS loading buffer and separated over a 12% Bis-Tris gel in
as the coupling reagents with 5-fold excess of each amino acid. MOPS buffer and stained with coomassie blue (Generon Cat.
Cleavage was performed by treatment of the peptide-resin with No. NB-45-00078-1L).
an ice-cold solution of (95% TFA, 2.5% H2O, 2.5% TIPS) for
30 min. Following ltration to remove the linker retaining resin,
ether was added to the ltrate. The peptide was ether soluble so
Conflicts of interest
following evaporation of ether the peptide was solubilised in There are no conicts to declare.
water and acetonitrile prior to lyophilisation. Aliquots of the
peptide were solubilised in a (2 : 1 : 1) mixture of H2O–MeCN–
MeOH (2 mL) and puried on a C8 reverse phase HPLC column
Acknowledgements
(Agilent PrepHT Zorbax 300SB-C8, 21.2 ꢂ 250 mm, 7 m) using We express our gratitude to Dr Geoff Kelly for recording some of
a linear solvent gradient of 15–60% (MeCN + 0.08% TFA) and the high eld NMR spectra and for his valuable assistance. NMR
(H2O + 0.08% TFA) over 40 min at a ow rate of 8 mL minꢀ1
.
spectra were recorded at the MRC Biomedical NMR Centre,
Fractions containing the product were combined and lyophi- Francis Crick Institute. This work was supported by the Francis
lised to dryness to give peptide 13 (4.4 mg, 6.7%). The puried Crick Institute which receives its core funding from Cancer
peptide was analysed by LCMS on an Agilent 1100 LC-MSD; Research UK (FC001029, FC001066, FC001999), the UK Medical
estimated purity 65%; MS (ES+) m/z: 468.0 [M + H]+, 489.9 [M Research Council (FC001029, FC001066, FC001999) and the
+ Na]+; calcd for C22H30N3FO7: 467.2.
Wellcome Trust (FC001029, FC001066, FC001999). This
SPPS of Z-AVLD(OMe)-FMK 14, Z-SAVLD(OMe)-FMK 15 and research was funded in whole, or in part, by the Wellcome Trust
Z-ASAVLD-FMK 16. The peptides were synthesised on an Intavis (FC001029, FC001066, FC001999). TUZ received funding from
ResPep SLi automated synthesiser (Intavis Bioanalytical the Boehringer Ingelheim Fonds. For the purpose of Open
Instruments AG, Cologne Germany), on a 0.1 mmol scale using Access, the author has applied a CC BY public copyright licence
© 2021 The Author(s). Published by the Royal Society of Chemistry
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Y. Hu, Z. W. Tao, J. H. Tian, Y. Y. Pei, M. L. Yuan,
Y. L. Zhang, F. H. Dai, Y. Liu, Q. M. Wang, J. J. Zheng,
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20464 | RSC Adv., 2021, 11, 20457–20464
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