Steps towards sustainable solid phase peptide synthesis: use and recovery ofN-octyl pyrrolidone

Article abstract of DOI:10.1039/d1gc00910a

The investigation of new green biogenic pyrrolidinones as alternative solvents toN,N-dimethylformamide (DMF) for solid phase peptide synthesis (SPPS) led to the identification ofN-octyl pyrrolidone (NOP) as the best candidate. NOP showed good performances in terms of swelling, coupling efficiency and low isomerization generating peptides with very high purity. A mixture of NOP with 20% dimethyl carbonate (DMC) allowed a decrease in solvent viscosity, making the mixture suitable for the automated solid-phase protocol. Aib-enkephalin and linear octreotide were successfully used to test the methodologies. It is worth noting that NOP, DMC and the piperidine used in the deprotection step could be easily recovered by direct distillation from the process waste mixture. The process mass intensity (PMI), being reduced by 63-66%, achieved an outstanding value representing a clear step forward in achieving green SPPS.

Full text of DOI:10.1039/d1gc00910a

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DOI: 10.1039/D1GC00910A
ARTICLE
Steps towards sustainable Solid Phase Peptide Synthesis: use and
recovery of N-octyl pyrrolidone.
Giulia Martelli,a Paolo Cantelmi,a Alessandra Tolomelli,*a Dario Corbisiero,a Alexia Mattellone,a
Antonio Ricci,b Tommaso Fantoni,a Walter Cabri,*a Federica Vacondio,c Francesca Ferlenghi,c Marco
Mor c and Lucia Ferrazzano.a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The investigation of new green biogenic pyrrolidinones as alternative solvents to N,N-dimethylformamide (DMF) for solid
phase peptide syntheis (SPPS) led to the identification of N-octyl pyrrolidone (NOP) as the best candidate. NOP gave good
performances in terms of swelling, coupling efficiency and low isomerization generating peptides with a very high purity.
The mixture of NOP with 20% dimethyl carbonate (DMC) allowed to decrease solvent viscosity making the mixture suitable
for the automated solid-phase protocol. Aib-enkephalin and linear Octreotide were successfully used to test the
methodologies. It is worth nothing that NOP, DMC and the piperidine used in the deprotection step could be easily recovered
by direct distillation from the process waste mixture. The process mass intiensity (PMI), being reduced by 63-66%, achieved
an outstanding value representing a clear step forward in SPPS greening.
common substitute, N-methylpyrrolidone (NMP)13 are reprotoxic
and were labeled as hazardous; accordingly, their replacement has
been considered “advisable or requested” by the ACS Green
Chemistry Institute® Pharmaceutical Round Table (GCIPR)14 that
defined this goal one of the hot topics in the list of the 12 green
chemistry key research areas (KRAs). The development of innovative
and general methods for sustainable amide bonds formation and
peptide synthesis has been also reported in the updated version of
the list of priorities.
On the basis of these considerations, selection of greener
alternatives needs to keep into consideration the sustainability and
the toxicity to natural environment and human health in the whole
solvent lifecycle.
Several solvents have been reported to be suitable for a greener SPPS
(GSPPS), such as, 2-methyl tetrahydrofuran (2-MeTHF),15 acetonitrile
(ACN) and THF,16 2-MeTHF and cyclopentyl methyl ether (CPME),17 -
valerolactone (GVL)18 and N-formylmorpholine (NFM),19 propylene
carbonate (PC),20 dimethylisosorbide (DMIE),21 and N-butyl
pyrrolidone (NBP).22 Green solvents mixtures affording proper
physicochemical properties for all the steps of solid phase processes,
Cyrene©/diethyl carbonate (DEC), sulfolane/DEC, anisole/dimethyl
carbonate (DMC),23 dimethyl sulfoxide (DMSO)/ethyl acetate
(EtOAc)24 and NFM/1,3-dioxolane (DOL)25 have been also explored.
However, some of these solvents like EtOAc, 1,3-dioxolane or 2-
MeTHF do not have the optimal characteristics for industrial
application because of their low flash point.
In this context, the family of N-alkylpyrrolidones may offer new
opportunities since, although having a similar structure to NMP, they
display a completely different metabolic profile, they are nor toxic or
reprotoxic and they still retain the main features of the parent
dipolar aprotic solvent. NBP has recently been applied by both
academia and industrial researchers to SPPS on polystyrene-based
resins, affording crude peptides with comparable quality to those
synthesized in DMF. In addition, NBP was also employed in mixture
with ethyl acetate providing interesting results.26
Introduction
The development of the solid-phase peptide synthesis (SPPS) that
originates from the seminal work of Bruce Merrifield back in the
sixties,1 gave access to previously unavailable long peptides via
iterative formation of amide bonds.2 Thanks to SPPS availability and
its automation, polypeptides became a target for medicinal chemists.
This modality is playing a major role in modern therapeutics,3 with
more than 70 peptides on the market and about >100 in clinical trial
advanced stages.
The different synthetic strategies for peptide synthesis have been
clearly described by Albericio in 2009.4 Since then, only two novel
industrial technologies have been reported, the enzymatic approach
by Enzypep5 and the hiving technology by Jitsubo.6 However, SPPS is
still the core technology for peptide synthesis and several research
groups are now focusing the attention on the optimization of every
single step to increase process greenness.7,8,9
Almost complete conversions are achieved thanks to the use of
reagents in excess that can be removed by extensive washing and
filtration of the solid support anchored growing peptide. Therefore,
in considering process greening, a fundamental role is played by the
solvents.10,11
N,N-dimethylformamide (DMF) is technically the perfect solvent for
SPPS. However, its toxicological profile12 is a serious matter of
concern in the pharmaceutical industry. In fact, DMF and its most
a. Department of Chemistry Giacomo Ciamician
Alma Mater Studiorum, University of Bologna
Via Selmi,2 40136-BO, Italy
E-mail: alessandra.tolomelli@unibo.it; walter.cabri@unibo.it
b. Fresenius kabi iPsum
via San Leonardo 23, 45010-RO, Italy
c. Department of Food and Drug Sciences
University of Parma
Parco Area delle Scienze 27/a, 43124-Parma, Italy
We have explored herein the characteristics of N-alkylpyrrolidones
and their use in SPPS, focusing our attention on safest members of
†
Electronic
Supplementary
Information
(ESI)
available.
See
DOI: 10.1039/x0xx00000x
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ARTICLE
Journal Name
this class as N-octyl-2-pyrrolidone (NOP), N-cyclohexyl-2-pyrrolidone biogenic GBL, but with a perspective of novel economically viable
(NCP) and N-benzyl-2-pyrrolidone (NBnP).
biomass treatment protocols, they might beDinOcIl:u1d0e.1d03iVn9ie/tDwh1eAGrtfCiuc0lteu0O9rne1l0iniAne
Aiming to the widest application, we have defined protocols for the list of biogenic solvents, displaying a high potential for more
manual or automatic SPPS, decreasing the PMI (product mass sustainable chemical approaches. In this context, since it will become
intensity) by solvent recycling.
inevitable to move away from a petroleum-based society, scientific
and technological efforts to turn these sustainable alternatives into
industrially feasible solutions are mandatory.
Results and discussion
N-Alkylpyrrolidones chemical-physical parameters, toxicological
data and in vitro metabolism
Table 2. Toxicity parameters of solvents evaluated in this study in comparison
with DMF and NMP33
In the selection of suitable candidates for DMF or NMP replacement
for GSPPS, chemical-physical parameters are the first characteristics
to be evaluated (Table 1).
N-alkylpyrrolidones share as a common feature the lack of acid
protons, while the elongation of the alkyl chain consistently
decreases their polarity moving from NMP to NBnP.
The high boiling point of this family of compounds, together with
their high flash point temperatures (always > 100°C, except for NMP),
are interesting characteristics for industrial application since there
are no safety concerns and solvents can be recovered by distillation.
Acute
Dermal
Toxicity
Acute Oral
Toxicity
(mg/kg bw)
Acute
Inhalation
Toxicity
Reproductive
Toxicity
Solvent
(mg/kg bw)
DMF
NMP
NBP
3010
150
> 5.8 mg/L air
> 5.1 mg/L air
> 5.1 mg/L air
n.a.
> 3160
> 5000
> 2000
> 4000
1600
Yes
Yes
No
300
NOP
NCP
> 2200
370
No
n.a.
No
NBnP
n.a.
n.a.
n.a.
n.a.
Table 1. Chemical-physical parameters of N-alkylpyrrolidonesa
n.a.: not available
Data reported in Table 2 clearly show that NOP, among the N-alkyl
pyrrolidones, is the safest one, being not reprotoxic and with very
high tolerability when administered orally or intradermally.
Viscosity at 25°C
Flash point
(°C)
Boiling point °C
(pressure mmHg)
Solvent
(mPa∙s)
DMF
NMP
NBP
0.8
1.65
4.0
58
36 (1)
91
68 (4)
The metabolism of these products generally shows oxidation at the
5 position of the lactam ring and on the first carbon of the N-alkyl
side chain. Through this pathway NMP generated consistent
quantities of the masked formaldehyde.34
108
142
141
113
80-85 (0.5)
114-115 (0.8)
94-97 (0.5)
124-125 (0.8)
NOP
NCP
6.6
11.5
21.4
NBnP
a See ref. 22a and 27
When NOP was incubated in the presence of rat (RLM) and human
liver microsomes (HLM), it was rapidly cleared with an average half-
life (t1/2) of 11.5 ( 2.0) min in RLM and 23.8 ( 5.6) min in HLM (Fig.
1).
Concerning N-alkylpyrrolidones’ viscosity, the observed values may
represent a challenge for large-scale application in automated
syntheses, since difficulties in transfer and purging may occur.
Anyway, a viscosity close to 4 mPA∙s is considered acceptable, if
efficiency of all the steps of SPPS (swelling, coupling, protecting
group cleavage) is maintained.9a In this context, the simple addition
of a low viscosity green cosolvent is a viable solution to overcome
this specific issue.
In the perspective of the industrial application of new SPPS protocols,
low cost, bulk availability and reliability of the supply chain of the
solvent has also to be ensured. Among the other pyrrolidones, NOP
is also known as SurfadoneTM LP-10028 and is widely used as a low-
foaming non-ionic rapid wetting agent and dynamic surface tension
modulator in cosmetic industry, pigment industry, cleaning
detergent production and polymer manufacturing. For this reason,
its price is comparable to that of the most common solvents (about
1-2$/kg). The industrial preparation of NMP is predominantly
derived from the reaction of γ-butyrolactone (GBL) with
monomethylamine.29 Similarly, other N-alkylpyrrolidones can be
prepared using the corresponding amines. Even if GBL is commonly
prepared via petrochemical route from succinic anhydride or succinic
acid, it could be obtained also by fermentation of sugar, and hence
be transformed from a fossil-based reagent to a renewable
source.30However, the cost of fermentations and the difficulties in
treating complex biomass-deriving matrices are still challenges to be
tackled31 and, even if the research on biorefineries is advanced,
technological implementation is hampered by a low profitability.32
Commercially available pyrrolidones are currently not prepared from
125
100
75
50
25
0
0
15
30
45
60
Time (min)
Fig.1 In vitro metabolic stability of N-octyl pyrrolidone (NOP). Time course of
NOP stability in rat (blue circles) and human (red circles) liver microsomes.
In vitro metabolism of NOP in RLM and HLM were very similar and
led to the formation of several metabolites (M1-M11, Fig.2, Fig. S1
and Table S1, Supporting Information). The major one was M1 (RT =
3.19 min; Fig. 2) having a m/z value of 212.1642, with a mass shift of
14 Da with respect to NOP. As shown in Fig.2 and Table S1, besides
M1, other minor metabolites were observed. A cluster of
metabolites (M6, M7, M9-11) displaying a mass shift M+16 with
respect to NOP, suggests a hydroxylation reaction (+O) while keto
2 | J. Name., 2012, 00, 1-3
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metabolites (M1, M2, M3, M4, M5, M8) displaying a mass shift M+14 before testing pyrrolidones in SPPS protocols, we firVsitelwy AertvicaleluOantleinde
(+O-2H) derive as second-generation metabolites from separately their efficiency in all these steps.DOI: 10.1039/D1GC00910A
dehydrogenation of hydroxylated ones. This is compatible with an
Solubility tests
oxidation reaction of a methylene to keto group involving either the
2-pyrrolidone ring or the N-octyl side chain of NOP. Low and High
energy mass spectra related to either keto or mono-hydroxylated
metabolites M1-M11 are reported in the Supporting Information as
Fig. S4-S14.
To evaluate the suitability of the selected solvents in SPPS, their
ability to solubilize commonly employed protected amino acids and
coupling reagents was investigated. Fmoc-Val-OH was initially
selected as the reference amino acid, demonstrating a perfect
solubilisation in all the tested solvents (NOP, NCP and NBnP) at a
standard 0.2 M concentration (Table 3). The same dissolution
protocol was applied to the main coupling reagent combinations:
OxymaPure®/DIC (A), COMU/DIPEA (B), PyBOP/DIPEA (C),
PyOxyma/DIPEA (D), HOBt/DIC (E), and HBTU (F) (Table 4). Optimal
results were generally achieved, except for HBTU (F), which was
scarcely dissolved in all the solvents and was hence excluded from
further studies. The solubility screening was then extended to Fmoc-
protected natural and non-natural amino acids (Fmoc-Gly-OH, Fmoc-
Ala-OH, Fmoc-Aib-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH and Fmoc-Thr(tBu)-
OH) that were dissolved at a 0.2 M concentration, both alone and in
the presence of the aforementioned additives after a 5 minutes pre-
activation (Table 3 and Table S2).
Solubility issues were detected in case of Fmoc-Cys(Trt)-OH, but
decreasing the concentration to 0.1 M, a slightly lower value than the
standard used in SPPS, good solubility was recovered in NCP, even if
not in NBnP (Table 3). When the amino acids were mixed with
coupling additives, only in sporadic cases the solubility was
incomplete, requiring an extra-dilution to observe clear solutions.
Fmoc-Cys(Trt)-OH demonstrated in this case a good solubility when
combined with OxymaPure®/DIC (A) and COMU/DIPEA (B) in NOP
and NCP, overcoming the above reported limited solubility of the
pure Fmoc-protected amino acid. On the other hand, NBnP was not
able to dissolve this amino acid, even in combination with coupling
reagents or in more dilute solutions (Table S2).
Fig. 2. HPLC-HRMS trace of a HLM incubation of N-octyl pyrrolidone (NOP) at
t=3h. Parent compound NOP and metabolites M1-M11 are reported together
with their RT. The most abundant metabolite (M1, with a mass shift of + 14
with respect to NOP) and two minor metabolites (M6 and M7, with a mass
shift of + 16 with respect to NOP) co-elute. Co-eluting metabolites are
detectable as Extracted Ion Chromatograms in Fig. S3, Supporting
information.
Effect of N-Alkylpyrrolidones as solvents in key steps of SPPS
Overall, OxymaPure®/DIC and COMU/DIPEA showed to be the best
coupling systems because of their general good results with all
solvents and amino acids (see Figures S16-S18 in Supporting
Information).
Solvents for SPPS need to fulfill a series of requirements at the same
time: good swelling efficacy for different resins, ability to completely
solubilize amino acids, coupling reagents and related by-products,
promotion of both coupling and deprotection steps. Consequently,
Table 3. Solubilisation efficacy of Fmoc-AA(PG)-OH amino acids in N-alkypyrrolidonesa
Fmoc-AA(PG)-OH
Solvent
NOP
Val
Gly
Ala
Aib
Phe
Pro
Tyr(tBu)
Thr(tBu)
Lys(Boc)
Cys(Trt)
NCP
0.1 M
0.1 M
NBnP
aSolubilisation monitored at 0.2M concentration unless 0.1M is specified. Legend: green=soluble; yellow=moderately soluble; red=insoluble. PG: protecting groups
Table 4. Solubilisation efficacy of coupling reagents A-F and Fmoc-Val-OH with A-E in N-alkypyrrolidonesa
Mixture Fmoc-Val-OH + coupling
reagents (A-E)
Coupling reagents
PyOxyma/
DIPEA
D
HBTU
OxymaPure®/DIC
COMU/DIPEA PyBop/DIPEA
HOBt/DIC
Solvent
Val+A Val+B Val+C Val+D Val+E
A
B
C
E
F
NOP
NCP
NBnP
aLegend: green=soluble; yellow=moderately soluble; red=insoluble
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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ARTICLE
To notice, OxymaPure®/DIC has been reported over the last ten years Noteworthy, the degree of swelling should not greatly overcome 4
as a high-efficient coupling agent able to reduce racemization with mL∙g-1 since a large volume of solvent would lead to reagents
performances overcoming those displayed by most of the common dilution. However, the swelling is considered acceptable if it
analogues.35
corresponds to ±30% of the measured volume for DMF.22
The measured swelling values of pyrrolidones and DMF are reported
in Table 5 as a mean value of analyses in triplicate.
Swelling tests
The first key step in SPPS is the choice of the resin and its swelling. A
suitable solvent should be able to properly swell different
commercially available resins, demonstrating the broadest
applicability. Polystyrene based resins (PS-resins) cross-linked with
1−2% of divinylbenzene are the most commonly used due to their
mechanical and chemical stability as well as for the low cost and bulk
availability, but also TentaGel-based resins, made of a polystyrene
(PS)/polyethylene glycol (PEG) network are often used for their
increased compatibility with polar solvents. PEG-based resins,
commonly known as ChemMatrix resins (CM), have recently received
great attention for the good performances reported in the synthesis
of long peptides. On these basis, cross-linked polystyrene (PS -
NOP and NCP afforded excellent swelling of PS-based resins,
commonly employed for industrial purposes and resulted
comparable to DMF in swelling the Wang linker. Moreover, better
results were observed with Trt-Cl resin. This behaviour suggests that
these solvents display a higher affinity to less polar resin matrixes.
Concerning 100% PEG-based resins, acceptable results were found
for CM-RinkAmide when NCP and NBnP were used, the last one
affording a 15% lower swelling in comparison to DMF. Overall, N-
alkylpyrrolidones cover a satisfying range of applicability since acid
terminating peptides can be prepared using PS-Wang or PS Trt-Cl
resin with NOP and NCP while amide peptides may be synthesized
using CM-RA resin with NCP or NBnP.
Merrifield), PEG-PS (TentaGel
– TG) and pure PEG-based
(ChemMatrix - CM) resins were considered for swelling with N-
alkylpyrrolidones. Moreover, different acid-sensitive linkers were
considered for each matrix: Wang and Trt-Cl linkers for obtaining the
final peptide as a C-terminal acid, and Rink Amide linker for a C-
terminal amide peptide. Details on bead size, loading and cross-
linking of the selected resins are reported in Table S3.
Coupling reaction in solution phase
The efficiency of pyrrolidones in promoting the coupling step was
first investigated in liquid phase. Accordingly, the synthesis of model
dipeptide17 Z-Phg-Pro-NH2 was performed in the selected solvents
(NOP, NCP, NBnP) and in DMF as benchmark reaction, screening
different coupling reagent combinations, and checking both the
conversion and the racemization degree (Table 6).
Measuring the racemization ratio is fundamental to predict the
efficiency of the solvent in SPPS couplings, and its inhibition is
necessary to avoid further purifications and low final yields.
Dipeptide Z-Phg-Pro-NH2 is particularly prone to racemization due to
the acidity of the benzylic α-proton in Phg.38 Moreover, proline reacts
slower than other amino acids and achieving a complete conversion
represent the main hurdle.
All the couplings were performed using (L)-amino acids and the crude
was analysed by HPLC-MS injection after 3 h, in order to determine
the dipeptide conversion and the racemization degree. The couplings
were performed with equimolar amounts of the two amino acids and
with a concentration of 0.125 M. The conversions were generally
good in all conditions; moreover, DIC/OxymaPure® system provided
only about 1% racemization in pyrrolidones (Table 6, entry 6, 11, 16),
less than what observed in DMF. PyBOP/HOBT/DIPEA and HOBT/DIC
combination afforded high level of racemization in all the solvents
tested, including DMF. COMU/DIPEA and PyOxyma/DIPEA showed
better values than the latter two combinations, but resulted
generally less effective than DIC/OxymaPure®.
Considering these data, together with the solubilisation screening of
coupling reagents and activated amino acids, DIC/OxymaPure® was
considered the best system for further investigations. This choice
was supported also by their very low cost, essential requirement for
SPPS use on industrial scale.
Recently Amadi-Kamalu et al.36 developed a model to predict resin
swelling in a given solvent, highlighting how the solvent-resin
interaction is a complex process especially as the swelling could be
related to physical parameters such as bead size and degree of cross-
linking. Anyway, experimental evaluation of swelling still represents
the most reliable method and reported protocols were applied for all
the solvent/resin combinations.20,37
Briefly, a dry resin sample in the desired solvent was allowed to
mildly shake in a graduated syringe for 30 minutes; after 5 minutes
of re-equilibration, solvent was removed under vacuum and the
volume increase of the resin was measured (mL∙g-1).
In general, solvents giving resin swellings higher than 4 mL∙g-1 are
considered good, values between 2 and 4 mL∙g-1 correspond to
moderately good solvents and swelling lower than 2 mL∙g-1 identifies
poor candidates.
Table 5. Swellinga of resins in DMF and pyrrolidones at RT after 30 mins
DMF
(mL∙g-1)
5.6
NOP
(mL∙g-1)
5.5
3.4
1.6
1.7
4.3
1.4
3.4
NCP
(mL∙g-1)
5.1
3.6
1.6
2.5
3.4
1.6
6.1
NBnP
(mL∙g-1)
2.3
1.6
1.6
1.3
4.0
1.6
6.7
Resin
PS-Wang
PS-Trt-Cl
PS-RinkAmide
TG-Wang
TG-RinkAmide
CM-Wang
CM-RinkAmide
3.2
3.2
6.0
6.2
4.4
7.8
Deprotection kinetic tests
aData reported are the average of triplicate measurements, see Table S4 in Supporting To develop a total DMF-free method, a solvent displaying excellent
Information.
behaviour in the deprotection step is a fundamental tool. In this
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context, the removal of Fmoc group was investigated in liquid-phase
to evaluate the effect of the solvent and avoid possible interference
of resin swelling or peptide aggregation issues that could occur in
solid-phase.39 Piperidine is the base of choice in Fmoc SPPS, due to
its role as fast deprotecting agents and dibenzofulvene scavenger.
Accordingly, tests were conducted using a 20% piperidine solution to
investigate deprotection kinetic, as commonly reported in SPPS
protocols.9a
Qualitative kinetic tests on Fmoc-Phe-OH deprotection were carried
out according to the method developed by Jad et al.40 and
deprotection rates were monitored by HPLC-MS at fixed time
intervals.
Fmoc-Phe-OH deprotection was also tested in DMF VaienwdANrticBlePOanslinae
comparison. Reaction was considered compDleOteI: a10t.d10is3a9p/Dp1eGaCra0n0c9e10oAf
the HPLC peak corresponding to Fmoc-Phe-OH; notably, the
reactions performed in all investigated pyrrolidones revealed
complete Fmoc removal in 2 minutes (see Figures S31-S32 in
Supporting information), perfectly in line with DMF performances.
SPPS in environmentally favourable solvents
After investigating the effect of N-alkylpyrrolidones as solvents in
liquid phase couplings, the study was extended to the SPPS of Aib-
enkephalin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-COOH/COONH2).
Aib residue insertion represents a harsh step because of its steric
hindrance that often leads to slow-rate coupling reaction and
consequently partial mis-incorporation.15 The amount of the des-Aib
sequences provides a measure of the efficacy of each pyrrolidone in
comparison to DMF or standard alternatives.
Table 6. Conversion (%) and racemization ratio (%) in the synthesis of Z-Phg-
a
Pro-NH2
O
H
O
NH2
H
N
O
A-E
Coupling reagent
0°C to rt, solvent
, 3h,
N
Cbz
OH
NH2
Cbz
N
+
N
H
A
B
C
D
: OxymaPure®/DIC
: COMU/DIPEA
: PyBOP/HOBt/DIPEA
: PyOxyma/DIPEA
: HOBt/DIC
Aib-enkephalin was manually synthesized on different resin/solvent
combinations according to the best swelling results: (i) PS-Wang and
PS-Trt-Cl resins with NOP and NCP; (ii) ChemMatrix-RinkAmide (CM-
RA) resin with NBnP. Treatment with TFA-based cleavage cocktails
afforded the peptide as acid at the C-terminal in case (i) and as amide
in case (ii). These conditions also allowed the simultaneous removal
of tert-butyl group in Tyr side chain. Peptides were assembled on
Fmoc-Leu-preloaded resins using 20% piperidine as Fmoc-
deprotection agent and DIC/OxymaPure® as coupling reagents. N-
alkylpyrrolidones were used for all the synthetic steps, including
washings. Double Fmoc-Aib-OH couplings were performed in all
syntheses, according to common literature procedures.15,16,17 HPLC
purities of the crudes after cleavage are reported in Table 7.
When the reaction was performed using NOP, excellent purities were
observed, providing about 98% of the target pentapeptide and <1%
of des-Aib byproduct on Wang resin (entry 1). Less than 3% impurity
was generated on TrtCl-resin (entry 2). Interestingly, these results
are consistently better than those obtained in the reference solvents
DMF and NBP, which provided a des-Aib amount >11% and >8%,
respectively (entries 5-6 and 7-8). NCP resulted comparable to DMF
in terms of pentapeptide purity, but lower amounts of des-Aib were
detected (entries 3-4). NBnP instead demonstrated a very low
efficiency since only 4.3% of Aib-enkephalin was detected, while des-
Aib tetrapeptide (16%) and other incomplete sequences
corresponding to the remaining 80% of the total were identified in
the crude (Entry 9).
O
E
Coupling
Reagent
Entry
Solvent
DL (%)b
Conversion (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
1.9
0.9
24.1
1.0
13.2
0.9
6.6
17.1
2.5
11.7
1.1
2.2
21.6
2.3
13.2
1.4
1.6
32.1
0.7
92
99
98
87
85
95
82
94
96
99
96
82
92
83
99
99
99
99
95
99
DMF
NOP
NCP
NBnP
10.0
aAll the reactions were performed pre-activating (3 min) Z-Phg-OH. Conversion and
racemization were calculated by HPLC. bDefined as (Z-D-Phg-Pro-NH2)/(Z-L-Phg-Pro-NH2)
× 100. The DL epimer was identified after synthesis of Z-D-Phg-Pro-NH2 where Z-D-Phg-
OH was used as a starting material.
As a consequence, NCP and NBnP were not further studied while
NOP was instead selected as an ideal greener alternative to common
solvents.
Table 7. HPLC purities for Aib-Enkephalin pentapeptide assembled on different resins and solvents.
Entry
1
2
3
4
5
6
7
8
Solvent
Resina
PS-Wang
PS-Trt-Cl
PS-Wang
PS-Trt-Cl
PS-Wang
PS-Trt-Cl
PS-Wang
PS-Trt-Cl
CM-RA
Pentapeptideb%
Des-Aib (%)
0.8
Other (%)
97.7
97.4
88.5
89.9
85.8
88.1
91.1
91.6
4.3
1.5
-
1.0
1.1
-
-
-
-
79.7
-
NOP
2.6
10.5
10.0
14.2
11.9
8.9
8.4
16.0
47.0
NCP
DMF
NBP
9
10c
NBnP
DMF
CM-RA
53.0
aPre-loaded Fmoc-Leu-resins were used; bDouble Fmoc-Aib-OH couplings; cfrom Ref.16
This journal is © The Royal Society of Chemistry 20xx
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Green Chemistry
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ARTICLE
Journal Name
View Article Online
DOI: 10.1039/D1GC00910A
Table 8. HPLC Purity of Linear Octreotide in NOP, NOP/DMC, DMF and NBP.
NOP/DMC 80:20
Manual SPPS
NOP/DMC 80:20
Automated SPPS
Compound
RRT
NOP
DMF
NBP
Cyclized N,O shift
Cyclized
TM-N,O shift 1
TM-N,O shift 2
TM+CO2
0.83
0.88
0.92
0.95
0.97
1.00
1.10
1.14
1.18
1.26
-
0.7
1.0
5.2
-
77.6
-
11.7
-
-
-
-
-
-
1.4
5.8
-
-
2.4
0.7
4.2
4.8
77.7
-
7.2
-
3.0
1.4
2.4
21.6
65.1
1.7
5.2
0.8
1.8
100
2.0
-
86.0
-
10.0
-
2.0
-
5.1
76.8
-
9.0
-
1.9
100
TM
TM+Boc
TM+tBu
TM+Boc2
TM+tBu2
3.8
100
Product purity (%)a
100
100
aHPLC purity calculated as sum of all target product adducts; RRT = relative retention time
Consequently, more sustainable conditions for SPPS were applied to alternatives for the recovery process, being very lipophilic. The final
the synthesis of linear Octreotide (H-D-Phe1-Cys2-Phe3-D-Trp4-Lys5- mixtures can be washed with water and then NOP recovered by
Thr6-Cys7-Thr8-ol), the final intermediate obtained by solid-phase distillation or it can be directly distilled in order to recycle all the
synthesis in the industrial manufacturing of the API Octreotide.
volatile chemicals. DIC cannot be recycled being unstable and almost
This peptide was one of the first biologically stable somatostatin completely transformed into 1,3-diisopropylurea during the coupling
analogues to be synthesized and used in oncology.41 The entire step.
protocol was performed with NOP as solvent and compared with Coupling streams and deprotection streams should be recovered
reference syntheses in NBP and DMF. Preloaded Fmoc-Thr(tBu)-ol- separately in order to avoid piperidine consumption by the excess of
Trt-PS resin was used, being highly suitable to the synthesis in NOP. Fmoc-amino acids used in the coupling steps. When the mixture
Although Octreotide is a medium-length peptide, frequent mis- NOP/DMC is used, due to the small difference between piperidine
incorporation of Cys7 residue occurs and for this reason the coupling and DMC boiling points (106 °C vs 90 °C at 760 mmHg), these two
step to introduce this residue was repeated twice in all the synthetic components are recovered in the same fraction and reused for
sequences. Standard 20% piperidine solution and DIC/OxymaPure® deprotection steps in further SPPS processes, after rebalancing the
reagents were employed for deprotection and coupling steps, required relative ratio.
respectively. HPLC purities of the crudes after cleavage are reported The results obtained by applying these protocols to the SPPS of linear
in Table 8. It is important to mention that all the detected species are octreotide in comparison with the DMF standard process are
related to linear Octreotide and hence precursors of the final cyclic reported in Table 9.
API. Des-Cys7 or other truncated sequences were never observed.
Table 9. Process Mass Intensity (PMI) and recovery in GSPPS
The excellent results obtained using NOP both in the synthesis of Aib-
enkephalin and linear Octreotide showed that its high viscosity value
(6.6 mPa∙s for NOP vs 4.0 mPa∙s for NBP and 0.8 mPa∙s for DMF) does
not affect the SPPS efficiency. The issue of viscosity anyway has to be
considered when moving from manual synthesis to automatic
synthesizers. Heating the reaction system had been previously
applied as a solution in SPPS in NBP.34 Instead, in order to decrease
NOP viscosity and to perform the reaction at automatic synthetizer
without heating, DMC (0.59 mPa∙s) was selected as co-solvent. In
fact, DMC proved to be a good cosolvent for SPPS.23 Addition of 20%
DMC to NOP allowed to decrease the viscosity down to 3.94 mPa∙s
(see Table S5), thus allowing the use of automated systems. After
having evaluated the swelling degree of NOP/DMC 80:20 in the PS
resins (see Table S4), the synthesis of linear Octreotide intermediate
was repeated using this sustainable solvents’ mixture both with
manual and automated protocols (Table 8). In both SPPS protocols
the final purity was comparable with the corresponding synthesis
carried out in DMF, NBP and NOP alone.
PMI
after
recovery
Waste
Stream
Recovery
(yield %)
Entry
Solventa
PMI
NOP (85)
Pipb (95)
NOP (85)
NOP (85)
DMC/Pipb(95)
Depr
Coupling
Depr
1
NOP
722
268
NOP/DMC
NOP/DMC
2
3
748
735
256
Coupling DMC (95)
NOP (85)
DMF
-
-
-
aThe wastes coming from coupling steps and from deprotection steps were distilled
separately. bPiperidine (Pip) involved in the formation of DBF-piperidine adduct was
subtracted from the total piperidine volume.
The percentage of waste related to the solvents involved in swelling,
coupling, deprotection and washing steps is very similar in all
different protocols (NOP 67.8%, NOP/DMC 69.0%, DMF 68.4%, see
Table S7 and Table S8). Concerning the synthesis performed in NOP
(entry 1), piperidine was easily recovered in 95% yield, considering
the maximum amount that could be collected. NOP was recovered in
the second fraction of condensed liquid in 85% amount. Under these
conditions, PMI value for the global process was more than halved
(268 with solvent/base recovery vs 722 without).
Solvent and base recycling
NOP and NOP/DMC proved to be greener alternatives to DMF,
however, in order to achieve a truly sustainable protocol, the Process
Mass Intensity (PMI) should be decreased by recovering the main
source of waste, namely the solvents (NOP or NOP/DMC) and the
piperidine used in the deprotection step. NOP offers several
6 | J. Name., 2012, 00, 1-3
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Page 7 of 10
Green Chemistry
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Journal Name
ARTICLE
When the green mixture NOP/DMC was used for the same synthesis
(entry 2), distillation of the deprotection streams under vacuum
allowed to distil, at 25 mmHg, DMC and piperidine together (95%
each) in a 1:0.86 ratio (V/V), as confirmed by 1H NMR (see Supporting
Information). Further, vacuum increase to 0.25 mmHg allowed to
recover NOP in 85% yield. Finally, the distillation under vacuum of
the coupling wastes allowed to recover in high yield both DMC (95%)
and NOP (85%), decreasing the total PMI from 748 (without
recovery) to 256. The final waste related to solvent is limited to 10%
for the process with NOP and to 8.7% when DMC/NOP were used.
OxymaPure® could be isolated from the solid residue following the
protocol reported by Rasmussen.24 However, this additional recovery
procedure increased the PMI and allowed to isolate OxymaPure® in
38% yield; therefore, in our protocol we did not considered coupling
agent recovery.
Experimental procedures
General Methods
View Article Online
DOI: 10.1039/D1GC00910A
Unless otherwise stated, all materials, solvents and reagents
were obtained from commercial suppliers, of the best grade,
and used without further purification. The solvents used were
of high-performance liquid chromatography (HPLC) reagent
grade. Specifically, Fmoc amino acids and resins were supplied
by Iris Biotech, Merck or Fluorochem. Coupling reagents were
purchased from Merck or Novabiochem. Piperidine was
supplied by Merck. DMF, other organic solvents, and HPLC-
quality acetonitrile (CH3CN) were purchased from Merck. Milli-
Q water was used for RP-HPLC analyses. NOP was purchased
Merck.
Solid-phase synthesis of the peptides was carried out manually
or using CSBio-CS136X peptide synthesizer (automated
syntheses).
A full evaluation of NOP compared to other solvents commonly used
in SPPS should comprise a complete lifecycle perspective of the
solvent impact. A quantitative comparison concerning the carbon
footprint between SPPS protocols in DMF and NOP cannot be
performed due to lack of literature data on NOP. Anyway, for a
qualitative estimation, since all the reactants and procedures in the
two SPPS protocols are the same, the attention can be exclusively
focused on the solvent carbon intensity (contribution to CO2
emission), considering the primary production and the disposal by
incineration or recycling. The production technologies of NMP and
NOP are very similar. Thus, the carbon footprint can be calculated
from the NMP’s one (4.22 kg CO2/Kg)42 by considering a 20-30%
increase in carbon content per kg due to the octyl chain and to the
higher boiling point. Therefore, the estimated range could between
5.06-5.49 kg CO2/kg. The carbon footprint related to DMF production
has been reported to be lower than NMP (3.57 Kg CO2/Kg).43
However, NMP recovery by double distillation decreases the carbon
footprint by 40%.42 On the other hand, NOP can be recovered by
single distillation and contribution of this process to carbon footprint
is consistently lower than the advantage arising from the reduced
amount of virgin solvent required. We can expect to have an impact
on NOP recycling on the carbon footprint similar to the one of NMP
(>40%).
HPLC-MS analyses were performed on Agilent 1260 Infinity II
system coupled to an electrospray ionization mass
spectrometer (positive-ion mode, m/z = 100–3000 amu,
fragmentor 30 V), using columns Agilent Zorbax-SB-C18 5 μm,
250 x 4.6 mm or Phenomenex Luna C18 5 μm, 250 x 4.6 mm;
temperature: 25 °C; injection volume: 10 µL, UV: 220 nm,
elution phases: H2O+0.08%TFA (mobile phase A) and
CH3CN+0.08%TFA (mobile phase B), flow: 0.5 mL/min or 1.0
mL/min. Chemstation software was used for data processing.
1H NMR spectra were recorded with an INOVA 400 MHz
instrument with a 5 mm probe. All chemical shifts were quoted
relative to deuterated solvent signals.
Distillations were performed with an Edwards RV3 vacuum
pump.
The viscosity was measured on CS10 Bohlin (Malvern)
rheometer in a titanium cylinder/steal cup (Bob-Cup C25)
configuration at temperature of 25°C controlled by Julabo MP
thermostat. The calibration of the instrument was previously
verified with an appropriate standard, namely 2,2,4-Trimethyl-
1,3-pentanediol monoisobutyrate (tabulated viscosity at 30C°
=9,96 mPa s).
In vitro metabolism of NOP in rat and human liver microsomes
A NADPH-generating solution (10 mM glucose-6-phosphate, 2
mM NADP+, 5 mM MgCl2 and 0.4 U/mL glucose-6-phosphate-
dehydrogenase) was prepared in 100 mM phosphate buffered
saline (PBS) pH 7.4. 15 µL of RLM or HLM preparation (final
protein concentration = 1 mg/mL) were added to 60 µL of co-
factors mix and 222 µL of PBS buffer. Samples were pre-
incubated under continuous stirring at 37 °C for 5 min, then 3
µL of stock solution of NOP were added to start the reaction.
The compound was incubated at the final concentration of 10
or 100 µM. Aliquots of RLM and HLM incubations were collected
at the beginning of the experiment (t=0 min) and at t=3 h,
enzymatic reaction was quenched by addition of a double
volume of CH3CN, samples were centrifuged (16,000 x g, 10 min,
4 °C) and the supernatant directly injected into the HPLC-HRMS
system for metabolite identification.
These considerations allow to rebalance the apparent disadvantages
of NOP in comparison with DMF. The comparable carbon footprint
combined with the health, environmental and safety data and the
future perspective of a sustainable production of NOP from
biomasses make this potential solvent a credible sustainable
alternative.
Conclusions
The evaluation of alternative green and safe pyrrolidones allowed to
identify NOP and NOP/DMC as the best solvents in term of yield and
selectivity for SPPS. To test the solvent performances Aib-enkephalin
and linear Octreotide were synthetized by SPPS producing the final
products in excellent purities using both manual and automated
procedures. The solvents and the piperidine could be easily
recovered decreasing the process PMI by 63% for NOP protocol and
66% for NOP/DMC one. Selecting green alternatives to dipolar
solvents for SPPS is an extensively investigated issue, but the
development of a methodology including solvents and reagents
recovery and recycle represents a step forward in the achievement
of a really green SPPS.
UPLC-HRMS conditions for in vitro NOP metabolite profiling
A Waters Acquity UPLC I-Class (Waters, USA) coupled to a
Waters V-Ion IMS qToF was employed for ion mobility-enabled
acquisitions in metabolite ID workflow. The V-Ion IMS qToF was
daily calibrated employing Waters Major Mix (Waters, USA) and
system performance were daily checked for accurate mass and
This journal is © The Royal Society of Chemistry 20xx
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Collisional Cross Section (CCS) accuracy and precision by
Journal Name
View Article Online
injecting system suitability test (SST) mixture composed of nine Coupling in solution phase: synthesis of DZO-PI:h1g0-.1P0r3o9-/NDH1GC00910A
2
QC standards. For UPLC separation, mobile phases A and B were Z-Phg-OH (0.2 mmol) was dissolved in a glass vial with 2.5 mL of
CH3CN and ultra-pure water both with 0.1% v/v formic acid and DMF or the selected pyrrolidone (NCP, NOP or NBnP). The
column was an Acquity UPLC CSH C18 (2.1 x 100 mm, 1.7 µm; desired coupling reagents combination (0.3 mmol) was then
Waters, USA). The linear gradient was: 0 min: 5%A; 0-15 min: 5- added (reagents A-E in Table 6: OxymaPure/DIC, COMU/DIPEA,
95%A; 15-16 min: 95%A; 16-17 min: 95-5%A; 17-20 min: 5%A. PyBOP/HOBt/DIPEA, PyOxyma/DIPEA, HOBt/DIC). After
5
Total run time: 20 min. Flow rate: 0.5 mL/min; injection volume: minutes of preactivation, H-Pro-NH2 (0.2 mmol) was added. The
2 µL; column temperature: 50 °C. Acquisition range: m/z = 50- solution was stirred at 0 °C for 1 h, then at room temperature.
300 amu. Instrumental parameters were set as follows: source After 3 h from the beginning of the reaction, an aliquot (80 μL)
temperature: 120 °C; desolvation temperature: 500 °C; source of the solution was diluted with 0.5 mL of a 1:2 CH3CN/H2O
gas flow: 20 L/h; desolvation gas flow: 800 L/h; capillary voltage: mixture and injected in HPLC-MS, in order to monitor the
0.8 kV (ESI+); cone voltage: 20 V; collision energy: low energy: 4 conversion and the racemization ratio. Selected
eV; high energy: 10-45 eV; reference mass: leucine enkephalin chromatograms are reported in the Supporting Information.
[M+H]+ m/z = 556.27658. The software UNIFI v.1.8.2 (Waters, For HPLC separation, mobile phases
A and B were
USA) was employed for data acquisition and processing.
H2O+0.08%TFA and CH3CN+0.08%TFA and column was an
Agilent Zorbax-SB-C18 5 μm, 250 x 4.6 mm. The linear gradient
was: 0 min: 80%A; 0-15 min: 80-60%A; 15-20 min: 60%A; 20-30
Solubility Tests
1 mL of the desired pyrrolidone was added to 0.2 mmol of either min: 60-80%A. Total run time: 30 min. Flow rate: 1 mL/min.
the protected amino acid/coupling reagent (pair of reagents) or
both protected amino acid and coupling reagent(s), and stirred Solid phase synthesis of H-Tyr-Aib-Aib-Phe-Leu-OH/ H-Tyr-Aib-
at room temperature until a clear solution was observed. In case Aib-Phe-Leu-NH2 (Aib Enkephalin)
of not complete dissolution (see red boxes in Tables 3,4 and S2), Aib-Enkephalin manual syntheses were carried out at room
an extra addition of solvent was added to reach a concentration temperature in glass syringes fitted with a polyethylene porous
of 0.1 M and to achieve total solubilisation, unless otherwise disc, connected to a vacuum source to remove excess of
specified.
reagents and solvents, by using 0.2 g of preloaded Fmoc-Leu-
resins. Specifically, preloaded PS-Wang and PS-Trt-Cl resins
(loading 1.1 mmol/g) were used for synthesis with NBP, NOP
Resin Swelling
Weighted resin (0.1 g) was introduced into a 5 mL syringe fitted and NCP, preloaded ChemMatrix-RinkAmide resin (loading 0.5
with a polypropylene fritted disc (the void volume of the tip and mmol/g) was used with NBnP. After swelling of the resin in 2 mL
the syringe was estimated to be 0.2 mL). The desired solvent or of the selected solvent (DMF, NBP, NCP, NOP or NBnP), Fmoc
mixture of solvents (2 mL) was added to the resin, which was protective group was removed by 20% piperidine in the
shaken for 30 minutes at room temperature. The swollen resin selected solvent (2 times ×2 mL, 15 min each), then the resin
was allowed to stand for further 5 minutes, then the solvent was washed with the selected solvent (3 times ×1.5 mL, 1 min
was removed under vacuum. The volume of the dry resin was each). Fmoc-Phe-OH, Fmoc-Aib-OH, Fmoc-Aib-OH, Fmoc-
then read and the swelling value was calculated from the Tyr(tBu)-OH (three-fold excess with respect to the loading of
following formula:
Degree of swelling (mL/g) = 100 × (volume of the swelled resin fold excess of the reagents with respect to the loading of the
- 0.2 mL)/0.1 g. resin) for 3 minutes and coupled to the resin for 60 minutes. In
the resin) were pre-activated by OxymaPure® and DIC (three-
All the measurements were performed in triplicate, data were case of both Aib residues in the sequence, the coupling of Fmoc-
reported as medium value. Triplicate measurements with the Aib-OH was repeated a second time. After each coupling step
related standard deviation values are reported in Table S4.
the Fmoc protective group was removed by treating the peptide
resin with 20% piperidine in the selected solvent (2 times ×2 mL,
15 min each), and the resin was washed with the selected
Fmoc cleavage kinetics in solution phase
Fmoc-Phe-OH (0.1 mmol) was dissolved in the desired solvent (3 times ×1.5 mL, 1 min each). After Fmoc cleavage of
pyrrolidone. Piperidine was added to the suspension in order to the N-terminal amino group, the peptide resin was further
achieve the desired concentration (20% base solution) in the washed with DCM (3 times ×2 mL, 1 min each) and dried under
final 1 mL deprotection mixture total volume. The reaction vacuum for 12 hours. Dry peptide resin was suspended in 5 mL
mixture was stirred at room temperature and samples of the of the mixture TFA/TIS/H2O (95/2.5/2.5 v/v/v) and stirred for 2
solution (20 μL) were taken at t=0 (before base addition) and h. The resin was filtered off, washed with TFA (1 time × 1mL, 1
after 2, 4, 6, 8, 10, 15 minutes. The samples were diluted with min) and diisopropylether (25 mL) cooled to 4 °C was added to
1.5 mL of CH3CN/TFA (1% v/v) and injected in HPLC-UV. the solution dropwise. The peptide was filtered and dried in
Reaction was considered complete at disappearance of the vacuo to obtain crude Aib-Enkephalin that was directly analysed
HPLC peak corresponding to Fmoc-Phe-OH. All kinetics resulted by HPLC-MS (Table 7). Related chromatograms and MS spectra
complete after 2 minutes from the reaction beginning, are reported in the Supporting Information.
independently from the employed solvent. Selected For HPLC separation, mobile phases
A and B were
chromatograms are reported in the Supporting Information.
For HPLC separation, mobile phases and
H2O+0.08%TFA and CH3CN+0.08%TFA and column was a
were Phenomenex Luna C18 5 μm, 250 x 4.6 mm. The linear gradient
A
B
H2O+0.08%TFA and CH3CN+0.08%TFA and column was an was: 0 min: 80%A; 0-15 min: 80-60%A; 15-20 min: 60%A; 20-30
Agilent Zorbax-SB-C18 5 μm, 250 x 4.6 mm. The linear gradient min: 60-80%A. Total run time: 30 min. Flow rate: 0.5 mL/min.
was: 0 min: 95%A; 0-15 min: 95-5%A; 15-20 min: 5-95%A; 20-22
min: 95%A. Total run time: 22 min. Flow rate: 1 mL/min.
8 | J. Name., 2012, 00, 1-3
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Green Chemistry
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Solid phase synthesis of H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys- NOP (85% yield). In the same way, coupling waste was distilled
View Article Online
Thr-ol (Linear Octreotide)
in two steps allowing the recovery of DMC first (95%), then of
DOI: 10.1039/D1GC00910A
Linear Octreotide manual syntheses were carried out at room NOP (85%) under higher vacuum.
temperature by using Fmoc-Thr(tBu)-ol-Trt-PS resin (0.2 g,
loading 1.1 mmol/g) in glass syringes fitted with a polyethylene
porous disc, connected to a vacuum source to remove excess of
reagents and solvents. After swelling of the resin in 2 mL of the
Author contributions
G.M. and P.C. equally contributed to the research. A.T., W.C., A.R.
selected solvent (DMF, NBP, NOP or NOP/DMC), Fmoc
protective group was removed by 20% piperidine in the
selected solvent (2 times ×2 mL, 15 min each), then the resin
was washed (3 times ×1.5 mL, 1 min each). Fmoc-Cys(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH,
Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH (three-fold
excess with respect to the loading of the resin) were pre-
activated by OxymaPure® and DIC (three-fold excess of the
reagents with respect to the loading of the resin) for 3 minutes
and coupled to the resin for 60 minutes. In case of the first
inserted Fmoc-Cys(Trt)-OH (Cys7 in the final sequence) the
coupling was repeated a second time. After each coupling step
the Fmoc protective group was removed by treating the peptide
resin with 20% piperidine in the selected solvent (2 times ×2 mL,
15 min each), and the resin was washed (3 times ×1.5 mL, 1 min
each). After Fmoc cleavage of the N-terminal amino group the
peptide resin was further washed with DCM (3 times ×2 mL, 1
min each) and dried under vacuum for 12 hours. Dry peptide
resin was suspended in 5 mL of the mixture TFA/TIS/1-
dodecanethiol (90/5/5 v/v/v) and stirred for 4 h. The resin was
filtered off, washed with TFA (1 time × 1 mL, 1 min) and
and M.M. designed research; G.M., P.C., A.M., L.F., D.C., T.F. and F.F.
performed experiments; A.T., W.C. and F.V. analyzed data and all
authors contributed to writing the manuscript.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We kindly acknowledge Alma Mater Studiorum University of
Bologna, University of Parma and Fresenius kabi for financial
support. We thank METCO srl (Monteveglio, Bologna, Italy) for
kind support.
Notes and references
1. B. R. Merrified, J.Am.Chem.Soc., 1963, 85, 2149.
2. B. L. Bray, Nat. Rev. Drug Discovery 2003, 2, 587.
3. B. G. de la Torre, F. Albericio, Molecules 2020, 25, 2293.
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diisopropylether (25 mL) cooled to 4 C was added to the
solution dropwise. The peptide was filtered and dried in vacuo
to obtain crude linear octreotide. HPLC purities calculated as
sum of all target molecule adducts are reported in Table 8.
Automated syntheses of linear Octreotide was performed at
room temperature with CSBio-CS136X peptide synthesizer
using the same conditions reported for manual protocol with
the only exception that the mixture NOP/DMC 80:20 was used
as solvent through all the steps (Fmoc cleavage, coupling,
washings).
Final global deprotection/cleavage on dry resin was carried out
analogously to the manual synthesis. Related chromatograms
and MS spectra are reported in the Supporting Information.
For HPLC separation, mobile phases
A and B were
H2O+0.08%TFA and CH3CN+0.08%TFA and column was a
Phenomenex Luna C18 5 μm, 250 x 4.6 mm. The linear gradient
was: 0 min: 80%A; 0-15 min: 80-60%A; 15-20 min: 60%A; 20-30
min: 60-80%A. Total run time: 30 min. Flow rate: 0.5 mL/min.
Solvent/base recycling
Deprotection stream waste (including washings) and coupling
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Green Chemistry
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10 | J. Name., 2012, 00, 1-3
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