A sustainable strategy for the assembly of Glypromate? and its structurally-related analogues by tandem sequential peptide coupling

Article abstract of DOI:10.1039/d0gc00143k

This work describes an improved and greener methodology of solution-phase synthesis for the preparation of Glypromate? (glycyl-l-prolyl-l-glutamic acid, GPE), a potent neuropeptide for applications in neurodegenerative conditions such as Huntington's, Parkinson's and Alzheimer's diseases. This protocol comprises the assembly of the perbenzylated form of Glypromate? [Cbz-Gly-Pro-Glu(OBn)-OBn (5)] froml-proline. Following a tandem sequential peptide coupling strategy, two chemoselective peptide bonds are formed without the need for purifying the intermediates ensuing a one-pot fashion synthesis. EcoScale score and E-factor were selected as the green metrics to assess the environmental impact of the preparation of tripeptide5using this protocol. After optimization and application of greener conditions, intermediate5was obtained with 95percent global yield and 99percent purity (NMR, HRMS, and rp-HPLC), with excellent final EcoScale score of 75 out of 100 and global E-factor of 1.8. Glypromate? is achieved by removing N- and C-protecting groups by hydrogenolysis using Pd/C as the catalyst in 98percent yield, avoiding chromatographic techniques. Moreover, the protocol ensures stereochemical integrity (determined by VT-NMR and rp-HPLC) and was also successfully applied for the preparation of structurally-related Glypromate? analogues with higher degree of molecular complexity compatible with functionalized amino acids with different side chains. For the first time a one-pot protocol for the assembly of tripeptides with the removal of protecting groups in the same reaction vessel is reported.

Full text of DOI:10.1039/d0gc00143k

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Page 1 of 28
Green Chemistry
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DOI: 10.1039/D0GC00143K
A Strategy for The Assembly of Glypromate® and Structure-
Related Analogues by Tandem Sequential Peptide Coupling
a,
b,c
a
Ivo E. Sampaio-Dias, * Luís Pinto da Silva, Sandra G. Silva,
d
a
Xerardo García-Mera, and José E. Rodríguez-Borges
aLAQV/REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of
Porto, R. Campo Alegre 697, 4169-007 Porto, Portugal.
bChemistry Research Unit (CIQUP), Department of Chemistry and Biochemistry, Faculty of Sciences
of University of Porto, R. Campo Alegre 697, 4169-007 Porto, Portugal.
cLACOMEPHI, GreenUPorto, Department of Geosciences, Environment and Territorial Planning,
Faculty of Sciences, University of Porto, R. Campo Alegre 697, 4169-007 Porto, Portugal.
dDepartment of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, E-
1
*
5782 Santiago de Compostela, Spain
Corresponding Author E-mail: idias@fc.up.pt.

Green Chemistry
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DOI: 10.1039/D0GC00143K
Abstract
This work describes an improved and greener methodology in solution-phase synthesis for the
®
preparation of Glypromate (glycyl-L-prolyl-L-glutamic acid, GPE), a potent neuropeptide with
applications in neurodegenerative conditions such as Huntington’s, Parkinson’s and Alzheimer’s
®
diseases. This protocol comprises the assembly of the perbenzylated form of Glypromate [Cbz-Gly-
Pro-Glu(OBn)-OBn (5)] from L-proline. Following a tandem sequential peptide coupling strategy, two
chemoselective peptide bonds are formed without the need of purifying the intermediates, in a one-pot
fashion synthesis. EcoScale score and E-factor were selected as the green metrics to assess the
environmental impact for the preparation of tripeptide 5 under this protocol. After optimization and
application of greener conditions, intermediate 5 was obtained with 95% global yield and 99% purity
(NMR, HRMS, rp-HPLC), with excellent final EcoScale score of 75 out of 100 and global E-factor of
®
1
.8. Glypromate is achieved by removal of N- and C-protecting groups by hydrogenolysis using Pd/C
as a catalyst in 98% yield, avoiding chromatographic techniques. Moreover, the protocol ensures
stereochemical integrity (determined by VT-NMR and rp-HPLC) and was also successfully applied for
the preparation of structure-related Glypromate® analogues with higher degree of molecular
complexity compatible with functionalized amino acids and with different side chains. For the first
time it is reported a one-pot protocol for the assembly of tripeptides with the removal of protecting
groups in the same reaction vessel.
®
Keywords: Glypromate , GPE, Neurodegenerative Diseases, Neuroprotection, One-pot Synthesis.

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Green Chemistry
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DOI: 10.1039/D0GC00143K
Introduction
Insulin-like growth factor type 1 (IGF-1) is a potent neurotrophic factor,[1],[2] widely distributed in
[3]
the mammalian central nervous system, which in brain tissues undergoes metabolization mediated by
acid-proteases producing two peptide fragments: the truncated peptide des-(1-3)-IGF-1 and the N-
®
terminal sequence of glycyl-L-prolyl-L-glutamic acid (GPE, also known as Glypromate , as depicted in
Figure 1).
O
NH2
N
H
N
CO2H
O
CO2H
Glypromate® (GPE)
®
Figure 1. Structure of Glypromate (GPE).
While des-(1-3)-IGF-1 is a potent neurotrophic factor by interaction with IGF-1 receptor, GPE does
[4]
not bind to this receptor nor has any growth promoting effect. However, it has been shown that GPE
3
is able to displace the binding of [ H]-glutamate to membranes of the anterior brain and hypothalamic
synaptosomes,[5],[6] suggesting a possible interaction with the NMDA receptors. Nevertheless, GPE
displays several regulatory actions and neuroprotection activity within the brain.[7] For example, it
[4]
stimulates the release of potassium-induced acetylcholine in the brain cerebral cortex, and dopamine
[8]
in the striatum. Exogenous administration of GPE prevents neuronal death induced by N-methyl-D-
aspartate (NMDA), quinolinic acid or 6-hydroxydopamine in different regions of the brain.[9],[5] To
date, the neuroprotection mechanism behind GPE’s bioactivity is not fully understood.[10]
In a study of 2009,[11] Burgos-Ramos and co-workers show that GPE exerts a neuroprotective effect
in vivo through the activation of protein kinases B (AKt), which play an important role in multiple
cellular processes such as apoptosis, cell proliferation, among others.[11] Moreover, GPE is known to
prevent the degeneration of three main phenotypes of striatum neurons (GABA-ergic neurons, and also
cholinergic and NADPHd interneurons) in an animal model of Huntington's disease. Other studies in
different experimental models showed that the administration of GPE exerts neuroprotective effects
after ischemic processes,[12] reducing apoptosis and inhibiting microglial proliferation, thus preventing
the loss of neurons after hypoxic ischemia.[4],[5] GPE’s neuroprotective effects are well documented in
animal models of neurodegenerative processes, such as Huntington’s, Parkinson’s, and Alzheimer’s
diseases.[5, 9, 13]

Green Chemistry
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Intravenous administration of GPE in patients who undergo coronary artery DbOyI: 1p0a.1s0s39/gD0rGaCft0i0n1g43K
CABG) surgery significantly reduces brain damage,[14] and prevent the decline in cognitive function
(
which is commonly seen during the early postoperative period.[15] Since there are no currently drugs
approved to reduce cognitive impairment after CARB surgery, the activity of GPE is of utmost interest
as a possible and valuable therapeutic candidate.[16]
Along with the therapeutically potential of GPE in several neurological conditions, Brimble’s [17]
and García-López’s[18] research groups have reported several neuroprotective GPE analogues. Many of
these peptidomimetics are based on chemical modifications at one or more GPE’s residues.[16] For
example, Brimble and co-workers described a GPE analogue in which N,N-dimethylation at the
glycine residue in GPE structure afforded a bioactive compound (N,N-dimethyl-Glu-Pro-Glu-OH) with
neuroprotective activity at 1 mM with a recovery value of 30-35% comparable to the parent peptide
GPE (recovery value from 25% to 40%) at the same concentration.[17b] By the other hand, chemical
modifications at the C-terminal, namely the conversion of the α-carboxylic acid in glutamate residue to
N,N-dimethylamide group retains neuroprotective activity with recovery value of 58% in survival
assay using striatal cells injured by okadaic acid in respect to the control (GPE) with recovery value of
3
0.5% at 1 mM concentration.[17c] In another study, this group show a neuroprotective compound in
which the side chain of glutamate residue is absent (replaced by glycine).[17a] This analogue showed
2
0% recovery after striatal cell survival post-apoptosis-induced injury.[17a]
Besides these analogues, Cacciatore and co-workers described the conjugation of (R)-α-lipoic acid
at the glycine residue of GPE affording a multifunctional codrug with antioxidant and neuroprotective
activities.[19]
Considering the potential of GPE and structure-related analogues in neurological conditions[16]
along with the intense research seeking for novel neuroprotective agents with enhanced
neuroprotection and stability profiles,[20] gram-scale preparation of GPE and structural-related peptides
becomes mandatory either to perform chemical modifications or study new formulations and
applications.
Peptides, such as GPE can be synthesized by solid-phase peptide synthesis (SPPS) or by
conventional solution-phase synthesis (SPS). Although SPPS is an effective strategy for the
development of lengthy peptides, this methodology commonly makes use of great excess of reagents
(
ca 5 equiv. per amino acid) [21] in the smaller academic scale and during early drug discovery,[22]
moreover it requires the use of highly polar solvents such as N,N’-dimethylformamide (DMF) to
ensure complete dissolution of all the reagents and promote the peptide coupling.[23] Besides DMF, N-
methyl-2-pyrrolidone and dichloromethane (DCM) are occasionally used.[24] However, all these
solvents are classified as hazardous solvents by CHEM21,[25] and its use by the pharmaceutical

Page 5 of 28
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industry is probable to be restricted by REACH (Registration, Evaluation, AuDOthI:o10r.i1z0a39t/iDo0nGC0a0n1d43K
Restriction of Chemicals).[26] Therefore, greener alternatives to the use of DMF in peptide synthesis is
mandatory.[24] In addition, SPPS requires specific equipment and resins, which constitute an additional
expense for the standard organic chemistry labs.[27]
In contrast, SPS is more adequate for medium to large-scale preparation of small peptides, allowing
the use of equimolar quantities of reagents, and thus making this approach suitable for the
development of eco-friendly and optimized protocols for the preparation of peptides of high interest.[28]
Trotter and co-workers have developed a straightforward methodology in SPS for the synthesis of
GPE, but this methodology involves multiple-step reactions and require the purification of all
intermediates.[17c] Sequential reactions in the same reaction vessel (tandem processes) might
beneficiate from environment-friendly strategies, avoiding the use and production of toxic substances,
and the possibility to build complex molecules with high stereoselectivity.[29]
In this work, we sought to contribute with the design of an alternative methodology to the synthesis
of GPE and structure-related analogues in SPS, hampered on the green chemistry principles.
Results and Discussion
The synthesis of GPE by SPS is well documented in literature by Trotter and co-workers.[17c] This
synthetic route relies on the preparation of perbenzylated intermediate 5 as the key intermediate
(Scheme 1). This precursor (5) is then readily converted into GPE by concomitant removal of both N
and C-terminal protecting groups by hydrogenolysis catalyzed by palladium on carbon (Pd/C).
Iterative approach to GPE via intermediate 5
O
NHCbz
NH
i
NH
ii
N
98%
CO2H
CO2Me
CO2Me
1
2
3
iii 82% (from 2)
GPE
O
O
NHCbz
CO2Bn
NHCbz
N
iv
100%
N
v
H
N
CO2Bn
94%
CO2H
O
5
4
Reagents and conditions:
(
(
(
(
(
i) SOCl2, MeOH, -5 ºC to rt, N2, 19 h;
ii) TEA, DCC, Cbz-Gly-OH, DCM, 0 ºC to rt, N2, 18 h;
iii) 1 M NaOH (aq), 1,4-dioxane, rt, 21 h;
iv) TEA, EtOCOCl, H-Glu(OBn)-OBn . TsOH, DCM, N2, 0 ºC to rt, 17 h;
v) H2, 10% Pd/C, 80:20 MeOH-H2O, rt, 18 h.
Scheme 1. Iterative synthesis of GPE in solution-phase via intermediate 5.[17c]

Green Chemistry
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This iterative approach starts with readily available L-proline (1), which is converteDdOtI:o10m.10e39th/Dy0lGCe0s0te14r3K
derivative 2 upon activation with thionyl chloride in methanol. The amino ester 2 is then coupled with
Cbz-glycine using 1,3-dicyclohexylcarbodiimide (DCC) and triethylamine (TEA) in DCM to afford
dipeptide 3 (Scheme 1). Treatment of 3 with 1 M NaOH using 1,4-dioxane as solvent leads to the
obtention of dipeptide 4 with a global yield of 82% (from 2). Dipeptide 4 is then activated in situ using
ethyl chloroformate to give the corresponding mixed anhydride (under basic conditions), and
subsequently treated with dibenzyl L-glutamate to provide perbenzylated tripeptide 5 as a single optical
isomer in 100% yield. The global yield reported from 1→5 is 80%, yet the process requires the
changing of different (hazardous) solvents, three chromatographic purifications, harsh conditions, and
an extensive overall time of the process (75 h, from 1→5). The final step in the sequence
(hydrogenolysis of tripeptide 5) is very effective, affording GPE in 94% yield after trituration in
diethyl ether (Et O).
2
After the successful proof-of-concept of this one-pot process,[30] we hypothesize the possibility to
run these isolated reactions in the same reaction vessel hampered on the fundamentals of green
chemistry,[31] in order to develop a tandem process based on a one-pot synthesis approach.
In some cases, workup and isolation of the intermediates are not always necessary. When multiple
reactions are conducted in a single reactor, without the need to isolate or purify the intermediates, it is
possible to achieve the so-called “pot-economy”.[32] This concept is more than a practical and cheaper
way to synthesize a target molecule – it represents a greener alternative to do it. The advantages are
clear: reduction of the amount of solvents, less waste production, and lower time associated to the
overall process. Furthermore, this approach obviates the necessity to isolate intermediates, alleviating
the labour required for the synthesis of a specific molecular target and reducing the intrinsic costs.
Therefore, a great advance in the synthetic route from 1 to 5 proposed by Trotter and co-
workers,[17c] would be achieved by preparation of dipeptide 4 without the need of protecting L-proline
(1), avoiding useless steps. Additionally, acylation of 4 with dibenzyl L-glutamate to afford
perbenzylated tripeptide 5 would benefit from in situ activation of 4 without isolation of this
intermediate, ensuing a one-pot protocol.
By examination of the retrosynthetic map for the preparation of intermediate 5 depicted in Scheme
2
, dipeptide 4 can be synthesized from fully deprotected L-proline (1) and the commercially available
preactivated glycine in the form of succinimidyl ester (Cbz-Gly-OSu) in presence of a tertiary amine.
At this point, there is no necessity to isolate intermediate 4 before coupling with dibenzyl L-glutamate,
H-Glu(OBn)-OBn, since a tertiary amine is also required in the next peptide coupling (in situ
activation) upon the addition of a coupling reagent to afford perbenzylated tripeptide 5.

Page 7 of 28
Green Chemistry
Coupling
in situ activation)
Tertiary amine
View Article Online
DOI: 10.1039/D0GC00143K
(
HCl
O
O
.
NHCbz
CO2Bn
NHCbz
H2N
CO2Bn
N
N
H
N
CO2Bn
CO2Bn
CO2H
O
5
4
H-Glu(OBn)-OBn
Deprotection
Hydrogenolysis)
Coupling (preactivated amino acid)
Tertiary amine
(
GPE
1
Cbz-Gly-OSu
Scheme 2. Retrosynthetic map for the preparation of GPE via intermediate 5 highlighting a one-pot
strategy.
Considering a tertiary amine is required in both strategies (in situ activation and peptide coupling of
preactivated amino acids), there is a synthetic advantage to carry out the these highly effective single
reactions in the same reactor,[29] ensuing a one-pot synthesis without the need to isolate intermediate 4,
as highlighted in Scheme 3.
O
NHCbz
Base-H
Base
Base
NH
Cbz-Gly-OSu
N
1
NHS
-
Step A
Base
-H
CO2
CO2
4
Coupling
Reagent
Tandem Sequential
Peptide Coupling
Step B
Base-H PXn
O
Urea
NHCbz
O
N
Base
NHCbz
N
H
H-Glu(OBn)-OBn . HCl
N
CO2Bn
O
N
N
O
CO2Bn
HOBt, Base-H Cl
O
N
5
Step C
Scheme 3. Tandem sequential peptide coupling process for the assembly of tripeptide 5.
To test the viability of this process for the preparation of tripeptide 5,[33] it was selected standard
conditions in SPS: DCM as solvent, TEA as tertiary amine and 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethylaminium tetrafluoroborate (TBTU),[34] as coupling reagent (entries 1-3, Table 1). All the
reactions were performed at room temperature.

Green Chemistry
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DOI: 10.1039/D0GC00143K
Table 1. Optimization of the one-pot protocol for preparation of tripeptide 5.
Time / h
Step
B
Tertiary Coupling
Yield
(5) / %
Entry Solventa
Purification
EcoScaleb
amine
Reagent
A
6
6
6
6
C
2
2
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
DCM
DCM
TEA
TEA
TBTU
TBTU
TBTU
TBTU
TBTU
TBTU
BOP
1
Ex + CC
Ex + CC
Ex + CC
Ex + CC
Ex + CC
Ex + CC
Ex + CC
Ex + CC
PP
86
95
93
72
93
91
91
90
95
47
52
51
35
46
50
48
59
75
0.5
0.5
0.5
DCM
TEA
EtOAc
EtOAc
EtOAc
EtOAc
EtOAc
EtOAc
TEA
TEA
12 0.5
12 0.5
12 0.5
12 0.5
12 0.5
DIPEA
DIPEA
DIPEA
DIPEA
PyBOP
PyBOP
a
5
0 mL solvent for 4 mmol of
L
-proline
CC: Column chromatography; Ex: Liquid-liquid extraction; PP: Precipitation
N-Hydroxysuccinimide (NHS) esters are widely used in peptide science due to their high reactivity
towards amines, relative stability to hydrolysis, and low toxicity.[35] Following the one-pot cascade
reactions depicted in Scheme 3, it was found that the coupling between L-proline and Cbz-Gly-OSu
(Step A, entry 1 of Table 1) proceeds smoothly at rt, with complete consumption of Cbz-Gly-OSu
after 6 h (TLC). The following steps B and C are critical, since the activation of the dipeptide
intermediate 4 (step B) and the aminolysis (step C) are intended to occur without removal of the
byproducts.
After in situ activation of 4 with TBTU and addition of H-Glu(OBn)-OBn it was detected a new
spot in TLC. Due to its high-water solubility, NHS, a byproduct formed in step A, can be easily
removed from the reaction media by simple aqueous extraction.[35] Therefore, after the workup
protocol (liquid-liquid extractions), the oil obtained was chromatographed. Gratifyingly, tripeptide 5
was isolated in 86% yield as a white solid (entry 1, Table 1). Changing the time reaction of steps B
and C (0.5 and 1 h, respectively, in entry 3 of Table 1), had no significative impact on the yield when
compared with the conditions used in entries 1 and 2 of Table 1.
Proven the feasibility of this tandem regioselective process, alternative approaches were considered
to upgrade this protocol under the twelve pillars of green chemistry and design a competitive synthetic
route for the preparation of GPE in SPS. To accomplish this goal, green substrates, eco-friendly
reagents and solvents as well as mild reaction conditions are compulsory.[36]
Regarding its importance for a laboratory scale study, the EcoScale score[37] was selected as the
green metric of choice to assess the environmental impact of this tandem process. EcoScale scores a
chemical process based on several aspects: yield, cost, safety, technical set up, the temperature and
time of reaction, workup and purification procedures. An ideal chemical process is characterized by

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high selective reactions which require as little steps as possible, and that make use ofDiOnI:e1x0.p10e3n9/sDi0vGeC0a0n1d43K
easily commercial available reagents.[38] The score obtained for a chemical process are considered
excellent if the EcoScale score is >75, acceptable if >50 and inadequate if <50.[39] The EcoScale score
ranges from 0 (totally failed reaction) to 100 (ideal reaction) and attributes penalty points to each of
the parameters that are subtracted from the ideal score of 100. EcoScale is a powerful tool to study a
chemical process and offers several advantages: easy score calculation, easy comparison of chemical
procedures and accounts with many aspects of environmental impact in its procedure assessment.[36]
All the EcoScale scores for the conditions tested were obtained from the parameters previously
described in literature,[37] using the EcoScale calculator (http://ecoscale.cheminfo.org/calculator). The
scores listed in entries 1-3 in Table 1 ranged from 47-52, which are in the borderline of an acceptable
synthesis. To improve the overall EcoScale score eco-compatible conditions and reagents were
explored for the synthesis of tripeptide 5.
Starting with the solvent, it was checked the possibility to change DCM, which is classified as
hazardous,[25] seeking for greener alternatives. Among the list of recommended solvents,[25] ethyl
acetate (EtOAc) was selected due to its low toxicity (EtOAc has been approved by the U.S. Food &
Drug Administration as a food additive),[40] and higher vapor pressure. However, when we attempted
replacing the solvent to EtOAc under the same conditions previously listed in entry 3 of Table 1, the
global yield dropped from 93 to 72% (entry 4 of Table 1). It was found that due to lower solubility of
1
in EtOAc, step A required more time for completion.
In fact, in the beginning of the reaction it is observed the formation of a suspension that progresses
into a clear solution after 12 h, delivering 5 in 93% yield (entry 5, Table 1). Steps B and C did not
require further optimization. However, despite the replacement of DCM by EtOAc and the excellent
overall yield obtained, the final EcoScale score was 46 (entry 5, Table 1), which is considered an
inadequate synthesis.[37]
To reduce the environmental impact of this process, it was envisioned the replacement of TEA by a
more convenient tertiary amine such as N,N-diisopropylethylamine (DIPEA), also known as Hünig’s
base.[34] This simple operation had no significant impact on the yield of 5, nevertheless the EcoScale
score was improved from 46 to 50 (entry 6, Table 1), resulting in an acceptable synthetic process.[37]
Despite TBTU is very effective as a coupling reagent with low epimerization, it is classified as
explosive,[41] displaying a great impact in the final Ecoscale score, and therefore it should be replaced.
Recently, other uronium coupling reagents based on HOBt and HABt such as HBTU, HCTU and
HATU were reported to cause anaphylaxis,[42] and therefore should be avoided. Combination of
benzotriazole derivatives with carbodiimides are also used for peptide coupling,[34, 43] however the use
of carbodiimides is incompatible with this one-pot protocol since they are known to react with the

Green Chemistry
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NHS (generated in the reaction) to form succinimidoxycarbonyl-β-alanine-hydroxysuDOccI:i1n0.i1m039id/De0GeCs0t0e1r4,3K
resulting in β-alanine insertions.[44] Likewise, ethyl cyano(hydroxyimino)acetate also known as
“
oxyma pure”,[45] which is considered as a green additive for peptide synthesis to replace HOBt/HOAt
requires the use of carbodiimides and therefore cannot be employed in this protocol.
Phosphonium salts such (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP, also known as Castro’s reagent),[34] are widely used as coupling reagents,
providing high coupling efficiency and low amino acid racemization. BOP has been used in the
preparation of GPE analogues.[18a] Despite the coupling efficiency demonstrated by BOP was
comparable with TBTU based on the yields obtained (entry 7, Table 1), the EcoScale score was
decreased to 48. BOP reagent is known to originate hexamethylphosphoramide (HMPA), a
carcinogenic byproduct, which may account for the lower score.[46]
To overcome this issue, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
(
PyBOP), a derivative of BOP that does not produce HMPA,[47] was used as coupling reagent. PyBOP
has been successfully applied in difficult peptide couplings without noticeable racemization,[48] in
contrast to other phosphonium reagents such as PyBrOP which is less effective in suppressing
epimerization.[48] Despite PyBOP is listed in the group of “least preferred” reagents for peptide
production (along with COMU) based on differential scanning calorimetry (DSC) studies, the average
major left limit onset is 121 °C, being therefore problematic only at high temperatures and larger
scales. Nevertheless, PyBOP display excellent properties that make it an appropriated candidate as
coupling reagent to be used in the early phase of drug development: high coupling efficiency, low
epimerization and safety/toxicity profile.[22]
By employing PyBOP the EcoScale score has considerably increased (59 out of 100) with no
significant effect on the global yield (entry 8, Table 1), which represents a satisfactory process.
Although DIPEA and PyBOP can also be employed in SPPS, the one-pot process uses practically
stoichiometric amounts, minimizing the waste of these reagents.
Chromatography techniques have also a dramatic impact on the final EcoScale score,[37] therefore
efforts were made to avoid chromatography without compromising the purity of the final product.
Bearing in mind that tripeptide 5 is a solid and that after the liquid-liquid extractions the TLC is very
clean, it was envisioned the precipitation of the desired peptide from the crude. Several solvents were
tested and Et O found the best solvent to induce precipitation. Crystallization was also easily attained
2
in a mixture of EtOAc/Et O 1:9, affording colourless needle-like crystals upon filtration and washings
2
with cold Et O. The perbenzylated tripeptide 5 was obtained in excellent yield (95%) and purity (99%,
2
NMR and HPLC (see ESI† for details). With this workup the global EcoScale score was significantly
improved, achieving 75 out of 100 (Table 1, entry 9), which indicate an excellent synthetic process.

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Environmental factor (E-factor) is a useful green metric to assess the environmeDnOtIa: 1l0.i1m039p/aDc0GtCo0f014a3K
chemical process.[49] A higher E-factor means more waste production and, consequently, a pronounced
negative environmental impact.[49] The ideal E-factor is zero. The synthesis of tripeptides by SPPS
display, in average, an E-factor score of 81.0,[50] which compares unfavourably with other competitive
available methodologies such as classical SPS or mechanochemical processes (ball-milling). These
two strategies display, in average, E-factor scores of 7.1 and 5.0, respectively.[50] This data agrees with
the global E-factor calculated for the classical synthesis of tripeptide 5 developed by Trotter (E-factor
=
6.3, see ESI† for details).[17c] The global E-factor for the assembly of 5 using this one-pot
methodology proves to be superior to all the other methodologies, scoring only 1.8.
Interestingly, tripeptide 5 exhibit cis-trans conformers (72:27) in DMSO-d at room temperature, as
6
demonstrated by variable-temperature NMR experiments (VT-NMR, Figure S29 of ESI†). The
-1
experimental energy barrier for cis-trans isomerization was found to be of 18.8 kcal mol , being the
cis conformers the most populated at room temperature. The cis-trans isomerization seems to be
associated to the prolyl residue as demonstrated by ROESY and further supported by in silico studies
(see Figures S30 and S31 of ESI† for more details).
In a previous work, our research group has found that TBTU interact with NHS to generate in situ
another coupling reagent, O-[N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU).
[30]
Regarding that NHS is formed as byproduct during step A, the possibility of a putative NHS-
PyBOP interaction upon the addition of PyBOP in step B was checked to study the stability of the
1
coupling reagent in presence of NHS. Therefore, a H-NMR experiment was performed using
equimolar quantities of these reagents in DMSO-d for 12 h (Scheme 4).
6
PF6
N
PF6
N
N
N
P
O
P
O
O
N
NHS
N
N
N
+
HOBt
N
- NHS
O
N
PyBOP
6
(
not detected)
1
Scheme 4. H-NMR study for the PyBOP-NHS interaction.
1
The data retrieved from H-NMR spectrum in this study excludes the formation of the hypothesized
intermediate 6 under this protocol (Figures S26 and S27 of ESI†), ensuring the stability of PyBOP in
the presence of NHS. It is worth mentioning that due to the presence of residual water associated with
DMSO-d it is possible to detect the hydrolysis of PyBOP (approx. 27%, note the lack of water typical
6
signal at 3.30 ppm in Figures S26 and S27 of ESI†).

Green Chemistry
Page 12 of 28
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After optimization of the synthetic protocol for perbenzylated tripeptide 5, GPE waDsOeI:a10s.i1l0y39o/Db0tGaCi0n0e1d43K
by hydrogenolysis using Pd/C in methanol/water in practically quantitative yield (Scheme 5),[17c] with
no need of chromatographic purification after filtration and precipitation in Et O (see experimental
2
section).
O
O
NHCbz
CO2Bn
NH2
N
N
i
H
H
N
CO2Bn
N
CO2H
9
8%
O
5
O
CO2H
GPE
Reagents and conditions:
i) Pd/C, H2 (50 psi), MeOH/H2O (1:1, v/v).
Scheme 5. Synthesis of GPE from perbenzylated tripeptide 5.
HRESIMS and HPLC experiments were performed to complement the spectroscopic data (NMR)
obtained for tripeptide 5. The results are shown in Figure 2. HRESIMS reveals the presence of two
+
+
peaks with m/z = 616.26893 and 1231.52941 a.m.u. ([M + H] and [2M + H] , respectively), which is
in agreement with the mass expected for tripeptide 5. The HPLC chromatogram (insert, Figure 2)
clearly advocate the success of the purification process from the crude mixture, showing a single peak
at t = 22.2 min (99.0% purity).
R
2
1
1
1
1
000
750
500
250
000
2000
1750
1500
1250
1000
750
2
2.2 99.3
Insert: rp-HPLC chromatogram
22.2 min
99%
7
5
2
50
00
50
0
500
250
20.0 0.7
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
Minutes
+
+
Figure 2. HRESIMS of tripeptide 5 ([M + H] = 616.26893 and [2M + H] = 1231.52941 a.m.u.).
Insert: rp-HPLC chromatogram. Purity of 99% (t = 22.2 min).
R

Page 13 of 28
Green Chemistry
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Considering the importance of GPE and its structural-related analogues, the synthDeOtI:ic10.1p0o39te/Dn0tGiCa0l0o14f3K
this protocol was also explored for the assembly of valuable GPE intermediates which may allow
subsequent functionalization either at the amino or carboxy termini sites. To accomplish this task, two
orthogonally protected GPE intermediates were designed: Cbz-Gly-Pro-Glu(OMe)-OMe and Boc-
Gly-Pro-Glu(OBn)-OBn (tripeptides 7 and 8, respectively, Scheme 6).
O
O
NHCbz
NH2
N
N
iv
H
N
H
CO2Me
N
CO2Me
8
8%
O
CO2Me
7
O
CO2Me
9
i
92%
CO2H
1
One-pot
Assembly and Deprotection
i, iv 70%
NH
v
75%
ii 89%
NHBoc
O
O
NH3
N
N
CF3CO2
iii
H
N
H
N
CO2Bn
CO Bn
2
9
6%
O
CO2Bn
8
O
CO Bn
10
2
O
NHBoc
N
iv
H
N
CO2H
91%
O
CO2H
11
Reagents and conditions:
i) Cbz-Gly-OSu, DIPEA, EtOAc, then PyBOP, H-Glu(OMe)-OMe
ii) Boc-Gly-OSu, DIPEA, EtOAc, then PyBOP, H-Glu(OBn)-OBn
iii) TFA, CH2Cl2
iv) Pd/C, H2 (50 psi), MeOH/H2O (1:1, v/v)
v) TFA (neat)
Scheme 6. One-pot synthesis of peptides 7-10 and preparation of GPE derivatives 9-11 in two-pots.
In the case of tripeptide 7 (Scheme 6), dimethyl L-glutamate was used instead of the benzylic ester
counterpart employed for derivative 5, delivering tripeptide 7 in 92% yield under the same one-pot
methodology. For the assembly of tripeptide 8 (Scheme 6), the appropriated Boc-protected glycine
preactivated as N-hydroxysuccinimidyl ester (Boc-Gly-OSu) was used instead of the Cbz amino ester,
delivering 8 in high yields (89%). Considering the lability of N-Boc protecting group under acidic
conditions,[30] the workup for tripeptide 8 was performed using NaHCO (aq).
3
With peptides 7 and 8 in hands it was performed the selective deprotection either at N or C-terminal
(Scheme 6). For tripeptide 7, selective removal of Cbz group was performed by hydrogenolysis
catalyzed by Pd/C, enabling the preparation of N-deprotected tripeptide 9 in 88% yield. Brimble and
co-workers have previously described that peptide 9 displays neuroprotective activity in striatal cell
survival post-apoptosis-induced injury.[17a]

Green Chemistry
Page 14 of 28
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In contrast, under the same conditions, selective hydrogenolysis of tripeptide 8DaOfI:f1o0r.1d03e9d/D0thGCe00C14-3K
deprotected tripeptide 11 in high yield (91%). In both cases, peptides 9 and 11 were induced to
precipitate in Et O, avoiding the need of chromatographic purifications.
2
Acidolysis of the carbamate 8 using trifluoroacetic acid (TFA), delivered N-deprotected tripeptide
1
0 as a trifluoroacetate ammonium salt in practically quantitative yield (96%) and with high purity
after the workup by precipitation in Et O.
2
In order to improve the potential of this protocol, the assembly and orthogonal deprotection were
carried out in the same reaction vessel. This unprecedented synthetic approach lead to the preparation
of peptides 9 and 11 directly from L-Pro. In the case of peptide 9, after the assembly of the
intermediate 7, the solvent was removed in vacuo and the N-protecting group (Cbz) was removed by
hydrogenolysis catalyzed by Pd/C without isolation of 7. The reaction proved to be feasible with 70%
yield for the global process, with the need of chromatographic isolation of tripeptide 9. In a similar
way, for the preparation of peptide 10, after the one-pot synthesis of intermediate 8, the solvent was
removed in vacuo, followed by the removal of Boc group by acidolysis upon the addition of TFA
(neat). The reaction delivered tripeptide 10 after chromatography with 75% global yield.
Modification on peptide side chains is commonly employed as a chemical strategy to tune
bioactivity. Moreover, many neuroprotective GPE analogues are composed by other amino acids with
more functionalities. In this sense, to ensure the compatibility of this one-pot methodology with the
preparation of GPE peptidomimetics it was designed and synthesized tripeptide 13 (Scheme 7). This
peptide contains a functionalized trans-4-hydroxy-L-proline (Hyp) and L-alanine residue as proline and
glycine surrogates, respectively (Scheme 7).
O
OH
O
4
i
66%
H2N
Cl
H2N
CF3CO2
12
CO2H
CO2H
H-L-Hyp-OH
NHCbz
ii 84%
O
N
H
O
N
CO2Bn
O
O
CO2Bn
4
13
Reagents and conditions:
i) Caproyl chloride, TFA
ii) Cbz-L-Ala-OSu, DIPEA, EtOAc, then PyBOP, H-Glu(OBn)-OBn
Scheme 7. One-pot synthesis of peptidomimetic 13.

Page 15 of 28
Green Chemistry
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Functionalized amino acid 12 was prepared by direct O-acylation of H-Hyp-ODHOI: 1w0.1i0t3h9/Dc0aGpCr0o0y14l3K
chloride in TFA as previously described in the literature.[51] The one-pot protocol was then applied to
amino acid 12 using Cbz-Ala-OSu and H-Glu(OBn)-OBn, affording tripeptide 13 in 84% yield. This
result shows that this one-pot methodology can be used for the preparation of GPE analogues with
higher degree of molecular complexity proving to be compatible with other amino acids.
Determination of the Stereochemical Purity using this Protocol
A paramount aspect when describing new methodologies employing chiral compounds such as
amino acids is the possible loss of optical activity. In peptide synthesis the activation step is critical,
and the most significant epimerization mechanism involves the transitory formation of
azlactones.[30],[34] Coupling reagents such as PyBOP, which contains the racemization suppressant
HOBt, are known to be very effective with minimal epimerization.[34] Azlactone formation is the major
mechanism that leads to loss of optical purity and generally occurs during the activation of carboxyl
groups of amino acids (except for glycine or proline).[30] Thus, this protocol is not expected to show
significative epimerization phenomena since activation takes place at proline and hence, is not prone to
undergo azlactone formation.[52] Furthermore, the mild conditions employed in this one-pot process,
the short overall time and the use of reagents in stochiometric amounts decreases the likelihood of loss
of optical activity.
Although the NMR data for the peptide products synthesized using this protocol (5, 7, 8 and 13)
show that these peptides are diastereomerically pure, further experiments were envisioned to
corroborate the spectroscopic data and unambiguously determine the diastereomeric ratio (dr).
Regarding that diastereoisomers may be resolved by simple chromatographic techniques such as rp-
HPLC, we prepared the mixtures of epimers for these peptides synthesized under the one-pot
methodology using D,L-proline instead of the optically active L-proline. These pairs of epimers were
then screened by rp-HPLC in order to access which pair suited better to access the diastereomeric ratio
(dr). Among the mixture of epimers tested, the pair 7/epi-7 were easily resolved by rp-HPLC. For that
purpose, both diastereopure peptides 7 and epi-7 (using L-proline or D-proline, respectively) were
prepared (Scheme 8).

Green Chemistry
Page 16 of 28
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DOI: 10.1039/D0GC00143K
O
NHCbz
D-Pro
4%
N
H
N
CO2Me
9
(R)
5
1.5 52.0 52.5 53.0 53.5 54.0 54.5 55.0 55.5
O
O
O
CO2Me epi-7
Minutes
O
NHCbz
N
i
L-Pro
2%
H
N
1
CO2Me
9
(S)
5
1.5 52.0 52.5 53.0 53.5 54.0 54.5 55.0 55.5
CO2Me
NHCbz
7
Minutes
O
D,L-Pro
7%
N
H
N
CO2Me
8
(
R/S)
5
1.5 52.0 52.5 53.0 53.5 54.0 54.5 55.0 55.5
CO2Me 7/epi-7
Minutes
Reagents and conditions:
i) Cbz-Gly-OSu, DIPEA, EtOAc, then PyBOP, H-Glu(OMe)-OMe.
Elution in gradient 1-40% acetonitrile in water, 80 min, flow: 1 mL min-1;
tR for 7: 53.7 min; tR for epi-7: 52.8 min.
Scheme 8. Epimerization assessment under the one-pot conditions for derivative 7 by rp-HPLC.
Using the one-pot protocol, tripeptides epi-7 and 7/epi-7 were obtained in 94% and 87% yields,
respectively, starting from the appropriate proline. The chromatograms obtained for each peptide along
with the NMR data for 7 and epi-7 (available in ESI) unarguably show that these peptides are virtually
free of epimerization (dr > 99%) by comparison with the NMR data and the chromatogram obtained
for the mixture of diastereoisomers 7/epi-7.
Conclusion
In this work is described a short, greener and effective strategy for the preparation of GPE via an
optimized tandem sequential peptide coupling reactions to assemble tripeptide 5 as the key
intermediate. This peptide is prepared in 13-14 h in contrast with classical procedures (75 h), with 90-
9
5% global yield and 99% purity (NMR, HRESIMS and rp-HPLC). Using VT-NMR experiments it
-1
was found a high energy barrier for cis-trans isomerization for tripeptide 5 (18.8 kcal mol ) in DMSO-
d6 which seems to be associated to the prolyl residue as demonstrated by ROESY and further
corroborated by in silico studies.
GPE is obtained from tripeptide 5 in gram-scale by simple hydrogenolysis using Pd/C as catalyst
with 98% yield, avoiding the need of chromatographic purifications. Additionally, it has been

Page 17 of 28
Green Chemistry
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demonstrated the broad applicability of this methodology through the preparation ofDOGI: P10E.103a9n/Da0lGoCg0u0e14s3K
using orthogonal protecting groups, enabling its N and C-functionalization. The assembly of
tripeptides and the removal of N-protecting groups in the same reaction vessel is unprecedented, as
demonstrated for preparation of peptides 9 and 10. Furthermore, this protocol allows the assembly of
peptides virtually free of epimerization (dr > 99%, rp-HPLC, NMR).
Although this methodology requires pre-activated amino acids (HOSu esters) for the success of the
tandem sequential peptide coupling process these amino acids are commercially available and/or are
easily activated using the NHS/DCC chemistry.
This highly efficient and competitive one-pot protocol offers a greener alternative for the gram-
scale preparation of GPE and structure-relates analogues with different side chains and functional
groups, boosting the development of novel neuroprotective peptides and the assembly of small
peptides in solution-phase.
Experimental Section
Chemistry. General
All chemicals were obtained from Bachem, Sigma-Aldrich and Fluorochem (reagent grade) and were
used without further purification. All reactions were carried out under argon atmosphere. Analytical
TLC was carried out on pre-coated silica gel plates (Merck 60 F , 0.25 mm) using UV light and an
2
54
ethanolic solution of phosphomolybdic acid (followed by gentle heating) for visualization. HPLC
analyses were performed on a Merck-Hitachi Lachrom Elite instrument equipped with a diode array
detector (DAD) and thermostated (Peltier effect) autosampler, using a Purospher STAR RP-18e
column (150 mm × 4.6 mm; particle size, 5 μm) and using the following conditions: 1–100% gradient
of B in A (A = H O with 0.05% of trifluoroacetic acid; B = acetonitrile) during 30 min with a flow rate
2
−
1
of 1 mL min and detection at λ = 220 nm (tripeptide 5); 1-40% gradient of B in A during 80 min with
−
1
a flow rate of 1 mL min and detection at λ = 220 nm (tripeptides 7, epi-7 and 7/epi-7); Mass spectra
were recorded with a LTQ OrbitrapTM XL hybridmass spectrometer (Thermo Fischer Scientific,
Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0 (Centro de Materiais da
Universidade do Porto, CEMUP). The capillary voltage of the electrospray ionization source (ESI) was
set to 3.1 kV. The capillary temperature was set at 275 °C. The sheath gas was set at 6 (arbitrary unit
as provided by the software settings). The capillary voltage was set at 35 V and the tube lens voltage
1
13
set at 110 V. H- and C-NMR spectra were recorded at CEMUP with a Bruker Avance III 400 at 400
MHz and 100 MHz, respectively. The NMR spectra were calibrated using residual protic solvents
[53]
(
D O: δ = 4.79; DMSO-d : δ = 2.50; 3.30, δ = 39.52) and are reported in ppm. The nomenclature
2 H 6 H C

Green Chemistry
Page 18 of 28
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used for the assignment of protons and/or carbons for each α-amino acid residue in thDeOIp: 1e0p.10ti3d9/eD0cGhCa0i0n14s3K
followed the three-letter system in subscript for amino acid residue (Glu: L-Glutamic acid; Gly:
Glycine; Hyp: trans-4-hydroxy-L-proline; Pro: L-Proline). The numeration of proton(s) and/or carbons
in the structures was performed starting with the carbonyl carbon of the main chain of each α-amino
acid residue as follows: H -X (for proton or magnetically equivalent protons) or C -X (for carbon);
AA
AA
AA: amino acid residue using three-letter system; X: proton(s) or carbon numeration starting from
carbonyl carbon of each residue of amino acid. In the case of presence of rotamers, the chemical shift
of the minor rotamer is assigned with “*”.
Dibenzyl N-(benzyloxycarbonyl)-glycyl-L-prolyl-L-glutamate (5)
In a round bottom flask previously flushed with Ar was charged H-L-Pro-OH (0.46 g, 4.0 mmol) in
EtOAc (50 mL), followed by the addition of DIPEA (1.39 mL, 7.98 mmol) and Cbz-Gly-OSu (1.23 g,
4
.02 mmol). The resulting suspension was stirred overnight after which a clear solution was achieved.
To this solution was added PyBOP (2.18 g, 4.19 mmol) with further stirring for 25-30 min until
complete dissolution, followed by the addition of H-Glu(OBn)-OBn hydrochloride (1.60 g, 4.40
mmol) and the solution was left stirring for 1 h. The suspension was transferred to a separatory funnel
and was washed with HCl 1M (2 x 50 mL) and NaHCO (2 x 50 mL). The organic extract was dried
3
over anhydrous Na SO , filtered and the solvent was removed in vacuo. The crude oil was either a)
2
4
chromatographed using EtOAc (2.22 g, 90% yield) or b) induced to precipitate by addition of cold
Et O/EtOAc (9:1) and filtrated (2.34 g, 95% yield), affording tripeptide 5 as white solid. Mp = 118-
2
23
[17c]
1
0
7
20 ºC [lit.[17c] 116-118 ºC]. [α] : ― 98.8 ± 0.1 (c1.06, CHCl ) [lit.
−55.9 (c0.37, CH Cl )]. R :
2 2 f
D
3
1
.61 in EtOAc. HPLC: > 99% purity (t = 22.3 min). H-RMN (DMSO-d , 400 MHz, 110 ºC) δ ppm:
R
6
.96 (br s, 1H, NH ), 7.60 – 7.07 (m, 15H, ArH), 6.62 (br s, 1H, NH ), 5.13 (d, J = 4.0 Hz, 2H,
Glu
Gly
CH Ph), 5.10 (s, 2H, CH Ph), 5.05 (s, 2H, CH Ph), 4.50 – 4.35 (m, 2H, H -2 + H -2), 3.84 (d, J =
2
2
2
Pro
Glu
4
.0 Hz, 1H, H -2), 3.80 (d, J = 3.5 Hz, 1H, H -2), 3.49 (t, J = 6.7 Hz, 2H, H -5), 2.46 (t, J = 7.6
Gly Gly Pro
1
3
Hz, 2H, H -4), 2.12 (tt, J = 15.9, 7.9 Hz, 1H), 2.70 – 1.70 (m, 5H). C-RMN (DMSO-d , 100 MHz,
Glu
6
1
10 ºC) δ ppm: [171.3 (C), 171.1 (C), 170.4 (C), 166.8 (C), C -1 + C -1 + C -1 + CO Bn], 155.5
Gly Pro Glu 2
(
C, NCO Bn), [136.6 (C), 135.7 (C), 135.4 (C), 3 x ArC], [127.7 (CH), 127.7 (CH), 127.6 (CH), 127.3
2
(
CH), 127.2 (CH), 127.1 (CH), 127.1 (CH), 127.0 (CH), 126.8 (CH), 3 x ArC], [65.5 (CH ), 65.0
2
(
CH ), 65.0 (CH ), 3 x CH Ph], [58.7 (CH), 51.0 (CH), C -2 + C -2], 45.6 (CH ), 42.4 (CH ), 29.5
2
2
2
Pro
Glu
2
2
+
+
(
CH ), 25.7 (CH ). HRMS (ESI-TOF) m/z: [M + H] calcd for C H N O 616.2653; found:
2
2
34 38
3
8
6
16.2689.

Page 19 of 28
Green Chemistry
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DOI: 10.1039/D0GC00143K
®
[17c]
Glycyl-L-prolyl-L-glutamic acid (Glypromate , GPE)
A mixture of tripeptide 5 (3.00 g, 4.87 mmol) and 10 wt % Pd/C (0.30 g) in 4:1 methanol–water
60 mL; v/v) was prepared in a hydrogenation vessel and transferred into a hydrogenation apparatus,
(
flushed with H (60 psi) and mechanically stirred at room temperature, protected from light, for 4 h.
2
The reaction mixture was filtered through a Celite™ pad and the pad washed with 1:1 methanol–water
(100 mL; v/v). The filtrate was concentrated to dryness under reduced pressure and the residue
triturated with anhydrous Et O to afford GPE as a white solid (1.39 g, 98%). mp: 215-216 °C
2
lit.[17c] 216 °C). [α] : ―61.6 ± 0.1 (c1.04, H O) [lit.
18
[17c]
26
1
(
[α] : ― 84.8 (c0.034, H O)]. H-RMN
D
D
2
2
(
D O, 400 MHz, 26 ºC) δ ppm: 4.51 (dd, J = 8.5, 4.1 Hz, 1H, H -2); 4.26 (dd, J = 8.8, 5.0 Hz, 1H,
2
P
r
o
H -2), 4.05 (d, J = 16.5 Hz, 0.80H, H -2), 4.00 (d, J = 16.5 Hz, 0.80H, H -2), 3.93* (d, J = 16.3
Glu
Gly
Gly
Hz, 0.20H, H -2), 3.76* (d, J = 16.3 Hz, 0.20H, H -2), 3.71 – 3.51 (m, 2H, H -5), [2.45 (t, J = 7.6
Gly
Gly
Pro
Hz), 2.43* (t, J = 7.6 Hz), 2H, H -4], 2.32 – 2.23 (m, 1H, H -3), 2.23 – 2.07 (1H, m, H -3), 2.07 –
Glu
Pro
Glu
1
3
1
.80 (m, 4H, H -3 + H -3 + H -4). C-RMN (D O, 100 MHz, 26 ºC) δ ppm: [179.4* (C), 179.3
Pro Glu Pro 2
(
C), C -5], [178.1 (C), 178.1* (C), C -1], [174.4 (C), 174.0* (C), C -1], [167.0* (C), 166.6 (C),
Glu Glu Pro
C -1], [61.5 (CH), 61.1* (CH), C -2], [55.4* (CH), 55.1 (CH), C -2], [48.5* (CH ), 47.8 (CH ),
Gly
Pro
Glu
2
2
C -5], [41.3 (CH ), 41.1* (CH ), C -2], [32.6* (CH ), 32.4* (CH ), 31.9 (CH ), 30.3 (CH ), C -4
Pro
2
2
Gly
2
2
2
2
Glu
+
C -3], [27.9 (CH ), 27.4* (CH ), C -3], [25.1 (CH ), 22.9* (CH ), C -4]. HRMS (ESI-TOF)
Pro 2 2 Glu 2 2 Pro
+
+
m/z: [M + H] calcd for C H N O 302.1347; found: 302.1348.
1
2
20
3
6
Dimethyl N-(benzyloxycarbonyl)glycyl-L-prolyl-L-glutamate (7)
Following the same one-pot protocol described for preparation of tripeptide 5, to a suspension of H-L-
Pro-OH (0.46 g, 4.0 mmol) in EtOAc (50 mL), was added DIPEA (1.39 mL, 7.98 mmol) and Cbz-Gly-
OSu (1.23 g, 4.02 mmol). To the resulting solution was added PyBOP (2.18 g, 4.19 mmol) and H-
Glu(OMe)-OMe hydrochloride (0.93 g, 4.4 mmol). After the typical workup the crude oil was
chromatographed using EtOAc as eluent affording the title compound as a colourless oil (1.71 g, 92%).
2
6
[17a]
1
[
α] : + 3.4 ± 0.2 (c1.31, CHCl ) [lit.
[α] : ― 62.3 (c0.23, CH Cl )]. H-RMN (DMSO-d , 400
D
D
3
2
2
6
MHz, 110 ºC) δ ppm: 7.89 (br s, 1H, NH ), 7.40 – 7.34 (m, 4H, ArH), 7.34 – 7.27 (m, 1H, ArH),
Glu
6
.65 (br s, 1H, NH ), 5.07 (s, 2H, CH Ph), 4.43 (dd, J = 8.4, 2.7 Hz, 1H, H -2), 4.34 (dt, J = 8.1, 5.7
Gly 2 Pro
Hz, 1H, H -2), 3.96 – 3.71 (m, 2H, H -2), 3.65 (s, 3H, CO CH ), 3.61 (s, 3H, CO CH ), 3.51 (t, J =
Glu
Gly
2
3
2
3
1
3
7
.0 Hz, 2H, H -5), 2.39 (t, J = 7.5 Hz, 2H, H -4), 2.15 – 2.01 (m, 2H), 2.01 – 1.83 (m, 4H). C-
Pro Glu
RMN (DMSO-d , 100 MHz, 110 ºC) δ ppm: [171.9 (C), 171.0 (C), 166.9 (C), C -1 + C -1 + C -
6
Gly
Pro
Glu

Green Chemistry
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1
+ CO Me], 155.6 (C, NCO Bn), 136.6 (C, ArC), 127.6 (2 x CH, ArC), 127.0 (CH, ArC), 126.9 (2 x
2 2
DOI: 10.1039/D0GC00143K
CH, ArC), 65.1 (CH , CH Ph), [58.9 (CH), 51.1 (CH), C -2 + C -2], [50.9 (CH ), 50.5 (CH ), 2 x
2
2
Pro
Glu
3
3
+
CO CH ], 45.7 (CH ), 42.4 (CH ), 29.3 (CH ), 25.7 (CH ). HRMS (ESI-TOF) m/z: [M + H] calcd
2
3
2
2
2
2
+
for C H N O 464.2027; found: 464.2030.
2
2
30
3
8
Dimethyl N-(benzyloxycarbonyl)glycyl-D-prolyl-L-glutamate (epi-7)
Following the same one-pot protocol described for preparation of tripeptide 5, to a suspension of H-D-
Pro-OH (0.46 g, 4.0 mmol) in EtOAc (50 mL), was added DIPEA (1.39 mL, 7.98 mmol) and Cbz-Gly-
OSu (1.23 g, 4.02 mmol). To the resulting solution was added PyBOP (2.18 g, 4.19 mmol) and H-
Glu(OMe)-OMe hydrochloride (0.93 g, 4.4 mmol). After the typical workup the crude oil was
chromatographed using EtOAc as eluent affording the title compound as a colourless oil (1.74 g, 94%).
2
3
1
[
α] : + 148.9 ± 0.2 (c1.065, CHCl ). H-RMN (DMSO-d , 400 MHz, 110 ºC) δ ppm: 7.71 (br s,
D
3
6
1
H, NH ), 7.39 – 7.33 (m, 4H, ArH), 7.33 – 7.27 (m, 1H, ArH), 6.67 (br s, 1H, NH ), 5.07 (s, 2H,
Glu Gly
CH Ph), 4.45 – 4.39 (m, 1H, H -2), 4.35 (dt, J = 8.3, 5.6 Hz, 1H, H -2), 3.88 (d, J = 15.7 Hz, 1H,
2
Pro
Glu
H -2), 3.86 (d, J = 15.6 Hz, 1H, H -2), 3.65 (s, 3H, CO CH ), 3.61 (s, 3H, CO CH ), 3.51 (t, J = 7.0
Gly
Gly
2
3
2
3
1
3
Hz, 2H, H -5), 2.37 (t, J = 7.5 Hz, 2H, H -4), 2.14 – 2.02 (m, 2H), 1.98 – 1.82 (m, 4H). C-RMN
Pro
Glu
(
DMSO-d , 100 MHz, 110 ºC) δ ppm: [171.9 (C), 171.0 (C), 167.1 (C), C -1 + C -1 + C -1 +
6 Gly Pro Glu
CO Me], 155.6 (C, NCO Bn), 136.6 (C, ArC), 127.6 (2 x CH, ArC), 127.0 (CH, ArC), 126.9 (2 x CH,
2
2
ArC), 65.1 (CH , CH Ph), [59.1 (CH), 51.1 (CH), C -2 + C -2], [50.9 (CH ), 50.5 (CH ), 2 x
2
2
Pro
Glu
3
3
+
CO CH ], 45.7 (CH ), 42.5 (CH ), 29.3 (CH ), 25.8 (CH ). HRMS (ESI-TOF) m/z: [M + H] calcd
2
3
2
2
2
2
+
for C H N O 464.2027; found: 464.2029.
2
2
30
3
8
Dimethyl N-(benzyloxycarbonyl)glycyl-D,L-prolyl-L-glutamate (7/epi-7)
Following the same one-pot protocol described for preparation of tripeptide 5, to a suspension of H-
D,L-Pro-OH (0.46 g, 4.0 mmol) in EtOAc (50 mL), was added DIPEA (1.39 mL, 7.98 mmol) and Cbz-
Gly-OSu (1.23 g, 4.02 mmol). To the resulting solution was added PyBOP (2.18 g, 4.19 mmol) and H-
Glu(OMe)-OMe hydrochloride (0.93 g, 4.4 mmol). After the typical workup the crude oil was
chromatographed using EtOAc as eluent affording the title compound as a colourless oil (1.61 g, 87%).
1
H-RMN (DMSO-d , 400 MHz, 110 ºC) δ ppm (mixture of epimers): 7.89 (br s, 2H, NH
+
Glu
6
NHGlu’), 7.46 – 7.34 (m, 8H, ArH), 7.33 – 7.26 (m, 2H, ArH), 6.66 (br s, 2H, NHGly + NHGly’), 5.07 (s,
4
3
4
H, 2 x CH Ph), 4.47 – 4.39 (m, 2H, H -2 + H -2’), 4.39 – 4.30 (m, 2H, H -2 + H -2’), 3.97 –
2 Pro Pro Glu Glu
.71 (m, 4H, H -2 + H -2’), 3.65 (s, 6H, 2 x CO CH ), 3.61 (s, 6H, 2 x CO CH ), 3.55 – 3.47 (m,
Gly
Gly
2
3
2
3
H, H -5 + H -5’), 2.41 – 2.34 (m, 4H, H -4 + H -4’), 2.18 – 2.02 (m, 4H), 2.01 – 1.83 (m, 8H).
Pro
Pro
Glu
Glu
1
3
C-RMN (DMSO-d , 100 MHz, 110 ºC) δ ppm (mixture of epimers): [171.9 (C), 171.8 (C), 171.1
6

Page 21 of 28
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(
C), 171.0 (C), 171.0 (C), 167.0 (C), 166.9 (C), C -1 + C -1’ + C -1 + C -1’ + C -1 + C -1’ +
Gly Gly Pro Pro Glu Glu
DOI: 10.1039/D0GC00143K
2
1
x CO Me], 155.6 (C, 2 x NCO Bn), 136.6 (2 x C, ArC), 127.6 (4 x CH, ArC), 127.0 (2 x CH, ArC),
2 2
26.9 (4 x CH, ArC), 65.1 (2 x CH , 2 x CH Ph), [58.9 (CH), 51.1 (CH), 51.1 CH), C -2 + C -2’ +
2
2
Pro
Pro
C -2 + C -2’], [50.9 (CH ), 50.5 (CH ), 4 x CO CH ], 45.6 (2 x CH ), 42.4 (2 x CH ), [29.3 (CH ),
Glu
Glu
3
3
2
3
2
2
2
+
+
2
9.3 (CH )], 25.7 (2 x CH ). HRMS (ESI-TOF) m/z: [M + H] calcd for C H N O 464.2027;
2 2 22 30 3 8
found: 464.2029.
Dibenzyl N-(tert-butyloxycarbonyl)glycyl-L-prolyl-L-glutamate (8)
Following the same one-pot protocol described for preparation of tripeptide 5, to a suspension of H-L-
Pro-OH (0.46 g, 4.0 mmol) in EtOAc (50 mL), was added DIPEA (1.39 mL, 7.98 mmol) and Boc-Gly-
OSu (1.09 g, 4.02 mmol). To the resulting solution was added PyBOP (2.18 g, 4.19 mmol) and H-
Glu(OBn)-OBn hydrochloride (1.60 g, 4.40 mmol). The suspension was transferred to a separatory
funnel and was washed with NaHCO (3 x 50 mL). The organic extract was dried over anhydrous
3
Na SO , filtered and the solvent was removed in vacuo. The crude oil was chromatographed using
2
4
2
3
EtOAc obtaining the title compound as a colourless oil (2.07 g, 89% yield). [α] : + 1.7 ± 0.1
D
1
(
c1.07, CHCl ). H-RMN (DMSO-d , 400 MHz, 110 ºC) δ ppm: 7.96 (br s, 1H, NH ), 7.43 – 7.27
3
6
Glu
(
m, 10H, ArH), 6.07 (br s, 1H, NH ), 5.14 (d, J = 3.7 Hz, 2H, CH Ph), 5.11 (s, 2H, CH Ph), 4.47 –
Gly 2 2
4
7
.37 (m, 2H, H -2 + H -2), 3.94 – 3.57 (m, 2H, H -2), 3.48 (t, J = 6.7 Hz, 2H, H -5), 2.46 (t, J =
Pro Glu Gly Pro
1
3
.6 Hz, 2H, H -4), 2.19 – 2.09 (m, 1H), 2.05 – 1.77 (m, 5H), 1.40 (s, 9H, Boc). C-RMN (DMSO-
Glu
d , 100 MHz, 110 ºC) δ ppm: [171.3 (C), 171.1 (C), 170.4 (C), 167.1 (C), C -1 + C -1 + C -1 +
6
Gly
Pro
Glu
CO Bn], 154.9 (C, Boc), 135.7 (C, ArC), 135.4 (C, ArC), 127.7 (2 x CH, ArC), 127.7 (2 x CH, ArC),
2
1
27.3 (CH, ArC), 127.2 (CH, ArC), 127.1 (2 x CH, ArC), 127.1 (2 x CH, ArC), 77.6 (C, Boc), 65.6
(
CH , CH Ph), 65.0 (CH , CH Ph), [58.8 (CH), 51.1 (CH), C -2 + C -2], 45.5 (CH ), 42.2 (CH ),
2
2
2
2
Pro
Glu
2
2
+
2
9.7 (CH ), 29.5 (CH ), 27.6 (3 x CH , Boc), 25.7 (CH ). HRMS (ESI-TOF) m/z: [M + H] calcd for
2 2 3 2
+
C H N O 582.2810; found: 582.2811.
3
1
40
3
8
Dimethyl glycyl-L-prolyl-L-glutamate (9)
Method A) Following the same protocol described for preparation of GPE, a mixture of tripeptide 7
0.50 g, 1.1 mmol) and 10 wt % Pd/C (0.10 g) in methanol (40 mL) was prepared in a hydrogenation
(
vessel and transferred into a hydrogenation apparatus, flushed with H (60 psi) and mechanically
2
stirred at room temperature, protected from light, for 4 h. After the typical workup the title compound
was obtained as light pink solid (0.32 g, 88%). Method B) Following the one-pot protocol described
for preparation of 7, after the addition of H-Glu(OMe)-OMe hydrochloride, the solvent was removed
in vacuo and the crude mixture was dissolved in methanol (40 mL) followed by the addition of

Green Chemistry
Page 22 of 28
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1
0 wt % Pd/C (0.10 g) and the reaction vessel was flushed with H (1 atm), at room temperature and
2
DOI: 10.1039/D0GC00143K
protected from light with magnetic stirring, for 4 h. The reaction mixture was filtered through a
Celite™ pad and the pad washed with 1:1 methanol–water (100 mL; v/v). The filtrate was
concentrated to dryness under reduced pressure and chromatographed with DCM:MeOH 20:1
2
3
affording the title compound as a light pink solid (0.25 g, 70%). [α] : ― 60.7 ± 0.2 (c1.075,
D
[17a]
1
[54]
CH OH) [lit.
[α] : ― 90.7 (c0.20, H O)]. H-RMN (DMSO-d , 400 MHz, 110 ºC) δ ppm : 8.13
D
3
2
6
(
br s, 1H, NH ), 4.48 (br s, 1H, H -2), 4.29 (dd, J = 13.7, 7.9 Hz, 1H, H -2), 3.92 – 3.67 (m, 2H,
Glu Pro Glu
H -2), 3.65 (s, 3H, CO CH ), 3.61 (s, 3H, CO CH ), 3.53 (t, J = 7.2 Hz, 2H, H -5), 2.40 (t, J = 7.5
Gly
2
3
2
3
Pro
1
3
Hz, 2H, H -4), 2.16 – 2.02 (m, 2H), 2.01 – 1.81 (m, 4H). C-RMN (DMSO-d , 100 MHz, 110 ºC) δ
Glu
6
ppm: [171.9 (C), 171.0 (C), 170.7 (C), 164.3 (C), C -1 + C -1 + C -1 + CO Me], [59.1 (CH), 51.1
Gly
Pro
Glu
2
(
CH), C -2 + C -2], [51.1 (CH ), 50.6 (CH ), 2 x CO CH ], 45.7 (CH ), 39.8 (CH ), 39.4 (CH ),
Pro Glu 3 3 2 3 2 2 2
+
+
2
3
9.5 (CH ), 25.6 (CH ). HRMS (ESI-TOF) m/z: [M + H] calcd for C H N O 330.1660; found:
2 2 14 24 3 6
30.1661.
Dibenzyl glycyl-L-prolyl-L-glutamate trifluoroacetate (10)
Method A) A solution of tripeptide 8 (0.52 g, 0.89 mmol) in TFA (2.05 mL, 26.8 mmol) was
magnetically stirred until consumption of the starting material (TLC). Excess of TFA was removed
using a rotavapor affording the title compound as a yellow oil (0.51, 96%). Method B) Following the
one-pot protocol described for preparation of 8, after the addition of H-Glu(OBn)-OBn hydrochloride,
the solvent was removed in vacuo and the crude mixture was dissolved in TFA (10 mL) and the
solution was magnetically stirred until consumption of the starting material (TLC). Excess of TFA was
removed using a rotavapor and the mixture was chromatographed with DCM:MeOH 20:1 to 10:1
2
3
affording the title compound as a yellow oil (0.40 g, 75%). [α] : ― 42.9 ± 0.2 (c1.135, CH OH).
D
3
1
[54]
H-RMN (DMSO-d , 400 MHz, 110 ºC) δ ppm : 8.09 (br s, 1H, NH ), 7.46 – 7.21 (m, 10H,
6
Glu
ArH), 5.14 (d, J = 5.1 Hz, 2H, CH Ph), 5.11 (s, 2H, CH Ph), 4.47 (d, J = 7.3 Hz, H -2), 4.41 (dd, J =
2
2
Pro
1
3.5, 7.5 Hz, H -2), 3.82 – 3.62 (m, 2H, H -2), 3.50 (t, J = 6.3 Hz, 2H, H -5), 2.47 (t, J = 7.5 Hz,
Pro Gly Pro
1
3
2
H, H -4), 2.23 – 1.73 (m, 6H, H -4 + H -4 + H -5). C-RMN (DMSO-d , 100 MHz, 110 ºC)
Glu
Pro
Glu
Glu
6
δ ppm: [171.3 (C), 170.7 (C), 170.4 (C), 164.0 (C), C -1 + C -1 + C -1 + CO Bn], [135.7 (C),
Gly
Pro
Glu
2
1
35.4 (C), ArC], [127.8 (CH), 127.7 (CH), 127.4 (CH), 127.3 (CH), 127.2 (CH), 127.2 (CH), ArC],
[
65.6 (CH ), 65.1 (CH ), 2 x CH Ph], [58.9 (CH), 51.2 (CH), C -2 + C -2], 45.6 (CH ), 39.4 (CH ),
2
2
2
Pro
Glu
2
2
+
2
9.7 (CH ), 28.4 (CH ), 25.7 (CH ), 23.3 (CH ). HRMS (ESI-TOF) m/z: [M + H] calcd for
2 2 2 2
+
C H N O 482.2286; found: 482.2282.
2
6
32
3
6
N-(tert-Butyloxycarbonyl)glycyl-L-prolyl-L-glutamic acid (11)

Page 23 of 28
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Following the same protocol described for preparation of GPE, a mixture of tripeptidDeO8I: 10(0.10.4398/Dg0G, C00.081343K
mmol) and 10 wt % Pd/C (0.10 g) in 4:1 methanol–water (40 mL; v/v) was prepared in a
hydrogenation vessel and transferred into a hydrogenation apparatus, flushed with H (60 psi) and
2
mechanically stirred at room temperature, protected from light, for 4 h. After the typical workup the
2
3
title compound was obtained as a hygroscopic white solid (0.30 g, 91%). [α] : ― 24.9 ± 0.1 (c1.21,
D
1
[55]
CH OH). H-RMN (DMSO-d , 400 MHz, 110 ºC) δ ppm : 7.77 (br s, 1H, NH ), 6.10 (br s, 1H,
3
6
Glu
NH ), 4.46 – 4.41 (m, 1H, H -2), 4.30 – 4.22 (m, 1H, H -2), 3.84 – 3.66 (m, 2H, H -2), 3.50 (t, J
Gly
Pro
Glu
Gly
=
6.8 Hz, 2H, H -5), 2.38 (t, J = 7.6 Hz, 2H, H -4), 2.20 – 1.83 (m, 6H, H -3 + H -3 + H -4),
Pro Glu Glu Pro Pro
1
3
1
1
.41 (s, 9H, Boc). C-RMN (DMSO-d , 100 MHz, 110 ºC) δ ppm: [172.9 (C), 172.0 (C), 170.9 (C),
6
67.1 (C), C -1 + C -1 + C -1 + CO H], 154.9 (C, Boc), 77.7 (C, Boc), [58.9 (CH), 51.1 (CH),
Gly
Pro
Glu
2
C -2 + C -2], 45.6 (CH ), 42.2 (CH ), 29.9 (CH ), 29.5 (CH ), 27.7 (3 x CH , Boc), 26.2 (CH ),
Pro
Glu
2
2
2
2
3
2
-
-
2
6.1 (CH ). HRMS (ESI-TOF) m/z: [M - H] calcd for C H N O 400.1725; found: 400.1729.
2 17 26 3 8
O-Caproyl-trans-4-hydroxy-L-proline (12)
In a round bottom flask was dissolved H-L-Hyp-OH (1.02 g, 7.78 mmol) in TFA (5 mL) at 0 ºC using
an ice bath, followed by caproyl chloride (2.20 mL, 15.7 mmol). The solution was stirred for 16 h and
the excess of TFA was removed in vacuo. To the resulting mixture was added cold Et O (50 mL) to
2
induce the precipitation. The precipitate was filtered and dried affording the title compound as a fine
white powder (1.76 g, 66%). The spectroscopic and spectrometric data is in agreement with the data
previously reported in the literature.[51]
Dibenzyl N-(benzyloxycarbonyl)-L-alanyl-(O-caproyl-trans-4-hydroxy)-L-prolyl-L-glutamate (13)
Following the same one-pot protocol described for preparation of tripeptide 5, to a suspension of 12
(
1.37 g, 4.00 mmol) in EtOAc (50 mL), was added DIPEA (1.39 mL, 7.98 mmol) and Cbz-Ala-OSu
1.29 g, 4.02 mmol). To the resulting solution was added PyBOP (2.18 g, 4.19 mmol) and H-
(
Glu(OBn)-OBn hydrochloride (1.60 g, 4.40 mmol). After the typical workup the crude oil was
chromatographed using EtOAc as eluent affording the title compound as a colourless oil (2.50 g, 84%).
2
4
1
[
α] : ― 66.0 ± 0.2 (c1, MeOH). H-RMN (CDCl , 400 MHz, 26 ºC) δ ppm: 7.43 – 7.27 (m, 15H,
D
3
ArH), 7.24 (d, J = 8.0 Hz, 1H, NH ), 5.72 (d, J = 7.6 Hz, 1H, NH ), 5.35 (br s, 1H, HHyp-4), 5.15 (s,
Glu
Gly
2
4
1
6
1
H, CH Ph), 5.08 (s, 2H, CH Ph), 5.07 (s, 2H, CH Ph), 4.67 – 4.53 (m, 2H, H -2 + H -2), 4.50 –
2 2 2 Hyp Glu
.36 (m, 1H, H -2), 3.78 (d, J = 2.9 Hz, 2H, HHyp-3), 2.50 – 2.16 (m, 7H), 2.03 – 1.93 (m, 1H), 1.66 –
Ala
.55 (m, 2H, CH̲ (CH2) CH ), 1.38 – 1.21 [1.25 (d, J = 6.7 Hz), 7H, H -3 + (CH ̲2 ) CH ], 0.89 (t, J =
2 2 3 Ala 2 3
1
3
.7 Hz, 3H, CH ). C-RMN (CDCl , 100 MHz, 26 ºC) δ ppm: [174.4 (C), 173.4 (C), 172.7 (C),
3
3
72.6 (C), 171.4 (C), 170.5 (C), C -1 + C -1 + C -1 + CO Bn], 155.7 (C, NCO Bn), [136.4 (C),
Ala
Hyp
Glu
2
2

Green Chemistry
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1
35.8 (C), 135.2 (C), 3 x ArC], [128.7 (CH), 128.6 (CH), 128.4 (CH), 128.3 (CH), 12DO8I.:210(.1C03H9/D),0G1C20801.143K
(
CH), ArC], 72.7 (CH, CHyp-4), [67.5 (CH ), 66.9 (CH ), 66.6 (CH ), 3 × CH Ph], 58.8 (CH, CHyp-2),
2
2
2
2
5
2.8 (CH , CHyp-3), [51.9 (CH), 51.8 (CH), CHyp-2 + C -2], 48.5 (CH, C -2), 34.2 (CH ), 33.6
2 Glu Ala 2
(
CH ), 31.3 (CH ), 30.0 (CH ), 27.3 (CH ), 24.5 (CH ), 22.4 (CH ), 18.1 (CH , C -3), 14.0 (CH ,
2 2 2 2 2 2 3 Ala 3
+
+
CH C ̲H ). HRMS (ESI-TOF) m/z: [M + Na] calcd for C H N NaO 766.3310; found: 766.3308.
2 3 41 49 3 10
Author Contributions
Conceived and designed the experiments: IES-D. Performed the experiments: IES-D. In silico
calculations: LPS. Analyzed the data: IES-D, LPS, SGS, XG-M, JER-B. Wrote the paper: IES-D. All
authors have given approval to the final version of the manuscript.
Supporting Information
1
13
1
D ( H, C, DEPT-135) and 2D (TOCSY, COSY, HSQC) NMR data for peptide products, free energy
calculations for the cis-trans isomerization of tripeptide 5, and the cartesian coordinates for cis-5 and
trans-5 conformers.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgments
The present work was supported by R&D projects UID/QUI/50006/2019 with funding from Fundação
para a Ciência e Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES,
Portugal) through national funds. LPS and SGS thank FCT for the Scientific Employment Stimulus
(
CEECIND/01425/2017 and CEECIND/01932/2017), respectively. Projects PTDC/BIA-
MIB/29059/2017, NORTE-01-0145-FEDER-000028, POCI-01-0145-FEDER-006980 and POCI-01-
145-FEDER-029059 are also acknowledged. IES-D thanks Mariana Andrade for the diligence with
0
NMR acquisition and Natália Tassi for the kind assistance with the HPLC experiments.
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Green Chemistry
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DOI: 10.1039/D0GC00143K
Graphical Abstract
H-Pro-OH
2
O
NH2
One-Pot Assembly and
Cbz-Gly-OSu
H-Glu(OBn)-OBn
N
H
3
Removal of Protecting Groups
1
N
CO2H
O
CO2H
O
NHR1
Glypromate®
N
H
R3
N
CO2R2
Tandem Sequential
Peptide Coupling
Deprotection
NHCbz
O
CO2R2
O
18.8 kcal mol-1
(DMSO-d6)
Glypromate Analogues
®
NHCbz
cis (major)
CO2Bn
O
trans (minor)
N
N
H
H
R1 = H, Cbz or Boc
R2 = H, Bn or Me
R3 = H or OCO(CH2)4CH3
N
N
CO2Bn
Yield: 90-95%
O
CO2Bn
O
CO2Bn trans-5
9
9% purity, >99% dr
PyBOP, DIPEA
EtOAc
EcoScale: 75; E-factor 1.8