Solid-Phase Peptide Synthesis Protocol

Research will be limited to:
- Method: Fmoc Solid-Phase Peptide Synthesis
- Material: Fmoc-protected amino acids
- Resin: Solid-phase peptide synthesis resin (unspecified type)
- Reagents: HATU, HBTU, DIC, Oxyma
- Analysis: UV Monitoring

Strategy: The objective is to synthesize a comprehensive Fmoc-SPPS protocol focusing on the comparative efficiency of coupling reagents (HATU vs. HBTU vs. DIC/Oxyma), resin selection criteria for difficult sequences, and specific handling of sensitive residues like Cys and Trp. I will search for literature comparing these specific coupling systems, protocols for UV-monitored deprotection to assess coupling efficiency, and optimized cleavage cocktails that prevent side reactions at Cys and Trp residues.
Queries:
- Fmoc-SPPS coupling reagents comparison HATU HBTU DIC Oxyma efficiency
- UV monitoring Fmoc deprotection kinetics peptide synthesis protocol
- SPPS resin selection guide swelling capacity loading density
- Fmoc-SPPS cleavage cocktail Cys Trp side reactions prevention
- double coupling strategy Fmoc-SPPS difficult sequences protocol

Executing 5 concurrent searches:
- Fmoc-SPPS coupling reagents comparison HATU HBTU DIC Oxyma efficiency
- UV monitoring Fmoc deprotection kinetics peptide synthesis protocol
- SPPS resin selection guide swelling capacity loading density
- Fmoc-SPPS cleavage cocktail Cys Trp side reactions prevention
- double coupling strategy Fmoc-SPPS difficult sequences protocol

Found 9 papers. Key excerpts:

Evaluation of combined use of Oxyma and HATU in aggregating peptide sequences — Andrea Caporale et al., 2017

Introduction

Polypeptides are finding increasing applications as therapeutics because of their specificity that often translates into excellent safety, tolerability, and efficacy profiles in humans . New synthetic methodologies for their preparation are thereby continuously sought to reduce the costs associated to chain assembly and purification. Solid-phase peptide synthesis (SPPS) is today a highly optimized and suitable technology to chemically produce peptides at various levels of complexity, including multiple cyclizations via canonical or innovative approaches and generation of peptidomimetics through introduction of unnatural amino acids and peptide bond surrogates, at both laboratory and industrial scale . Despite the outstanding progresses in the development of ever more efficient reagents and methods for acylation, deprotection, or cleavage reactions, aggregation phenomena occurring on the growing polypeptide chain and steric hindrance of particular derivatives used for amino acid incorporation still cause incomplete reactions that lead to the accumulation of a largely heterogeneous population of internally or terminally deleted fragments. The occurrence of so many side products and the intrinsic hydrophobicity that usually accompanies highly aggregating peptide fragments make the isolation of the target molecules an almost prohibitive or otherwise very expensive task. Kent and coworkers made a systematic study and classified difficult couplings as random (caused by slow reactivity of certain activated amino acids, commonly b-branched amino acids) and nonrandom (caused by side-chain aggregation phenomena due to the b-sheet secondary structure formation). In the case of nonrandom difficult couplings, incomplete amino acid incorporation is not generally prevented by following approaches often applied for difficult random couplings, such as prolonged reaction time, recoupling, and/or changing synthetic strategy .
In these cases, physical alterations of the reaction environment, such as using different solvents, addition of chaotropic agents, heating and use of microwaves that swell the solid support, favor molecular collisions, and disrupt non-covalent interactions occurring intermolecularly and intramolecularly, may be more beneficial . Among these tricks, microwave-assisted methods are particularly useful for the synthesis of peptides prone to form b-sheet-like structures and for improving poor yields associated to sterically hindered derivatives .
In addition, lowering resin loading, the introduction of pseudoprolines, and extension of N a -deprotection times have been proposed and in many cases successfully applied for difficult peptide synthesis . Exploring different efficient coupling reagents is also

an option in these cases, as they can improve coupling efficiency by maximizing the number of productive molecular collisions. Among the coupling agents more extensively used in peptide synthesis, we find several 1-hydroxybenzotriazole-based or 1-hydroxy-7-azabenzotriazole-based reagents including HATU , HBTU/TBTU, and HCTU/TCTU or phosphonium salts (PyAOP, PyBOP) [12,13]. Unfortunately, many of these are classified under a class 1 explosive category or have high cancerogenicity potentials and features that, for safety reasons, have an impact on costs increase . In the last years, a renewed interest has been devoted to the use of OxymaPure (r) , hereafter only Oxyma, as an alternative to benzotriazoles because of its compatibility with greener solvents and an improved balance between reactivity, solubility, and stability in various solvents . This reagent is also largely used as an effective epimerization-suppressing additive . Moreover, Albericio's group showed the high performance of Oxyma in association with COMU (1-[(1-(cyano-2-ethoxy-2-oxoethylidene-amino-oxy)-dimethylamino-morpholino-methylene)]- methan-aminium-hexafluorophosphate), a uronium-type coupling reagent, in microwave-assisted SPPS .
Despite the great potential of coupling reagents based on ethyl2-cyano-2-(hydroxyimino)acetate, a systematic study on its use in combination with other coupling reagents in SPPS is still lacking. In this frame, here we have investigated the use of Oxyma and HATU for the preparation of some peptides known as models of difficult synthesis. We have observed that double sequential couplings with Oxyma/DIC and HATU/Sym-collidine lead to largely improved yields in some difficult synthetic steps, thus affording final products that can be more easily isolated and characterized. On this basis, we report here a comparative study of the use of sequential double couplings with HATU/Sym-collidine or HATU/Symcollidine combined to Oxyma/DIC to improve the efficiency in the synthesis of some bioactive polype

ptides chosen from those used in our laboratories in ongoing projects and for which we have experienced synthesis problems (Ab , Prep1 , AIF , p1-27N-CHCHD4, hCFC ) or selected as models of difficult sequences (a-Syn , Aib-Enkephalin) [23,15].

Materials and Methods

Materials

All chemicals were from commercial sources and used without further purification unless otherwise stated. Solvents, including acetonitrile (CH 3 CN), dimethylformamide (DMF), dichloromethane, tetrahydrofuran, and methanol (CH 3 OH), were purchased from ROMIL (Dublin, Ireland). Other products such as Sym-collidine, N, N-di-isopropylethylamine, piperidine, acetic anhydride (Ac 2 O), and trifluoroacetic acid (TFA) were from Sigma-Aldrich (Milan, Italy). Protected amino acids and coupling agents HATU (1-[bis (dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3oxid hexafluorophosphate), Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate), and N-N 0 diisopropylcarbodiimide (DIC) used for peptide synthesis were from IRIS Biotech GmbH (Marktrewitz, Germany).

In silico prediction of peptide's aggregation potential

For each sequence, except Aib-Enkephalin, we evaluated the aggregation potential using two distinct open-source computational prediction tools: AGGRESCAN and Peptide Companion [24,25]. AGGRESCAN considers the experimentally calculated aggregation propensity value of each amino acid in a given sequence and identifies the hot spot regions as aggregation-prone segments. On the contrary, the software Peptide Companion evaluates the amino acid composition and predicts difficult coupling steps during the synthesis (see the Supporting Information section for more details).

MS characterization

ESI-TOF-MS analyses were performed with an Agilent 1290 Infinity LC System coupled to an Agilent 6230 TOF LC/MS System (Agilent Technologies, Cern

Suppression of alpha-carbon racemization in peptide synthesis based on a thiol-labile amino protecting group — Yifei Zhou et al., 2023

Introduction Solid-phase peptide synthesis (SPPS), which was developed by Nobel laureate R. Bruce Merrifield in the early 1960s 1 , 2 , is the most commonly used method for preparing peptides 3 , 4 , 5 . Protection of the α-amino group of the amino acids is key to the success of SPPS. The 9-fluorenylmethoxycarbonyl (Fmoc) and tert -butyloxycarbonyl (Boc) groups are widely used for this purpose, and SPPS processes that use these groups are referred to as Fmoc SPPS and Boc SPPS, respectively (Fig. 1a ) 6 , 7 , 8 . The former is simpler and more reliable than the latter and thus is used more often. Fig. 1: α-Amino protecting groups used for solid-phase peptide synthesis (SPPS). a Boc SPPS and Fmoc SPPS. b Synthesis of DNPBS-Cl ( 2 ). c Protection of benzylamine (BnNH 2 ) by DNPBS to afford 3 . d Removal of DNPBS from 3 . e UV absorption spectra of 3 and 4 in THF and pyridine. DNPBS, 2,4-dinitro-6-phenyl-benzene sulfenyl. Full size image Both Fmoc- and Boc-protected amino acids have a carbamate moiety attached to the α-C. Activation of the protected amino acid by the coupling reagent generates a racemizable intermediate during peptide bond formation and thus leads to α-C racemic products that are hard to remove by routine purification methods (Fig. 1a ) 9 , 10 . In addition, Boc and Fmoc are removed by treatment with a strong acid (trifluoroacetic acid [TFA]) or base (piperidine), respectively, and these harsh deprotection conditions lead to undesirable side reactions, such as aspartimide and piperidide formation 10 , 11 , 12 . Here, we report a thiol-labile amino protecting group, the 2,4-dinitro-6-phenyl-benzene sulfenyl (DNPBS) group, for SPPS. The use of DNPBS greatly suppresses the main side reactions observed in conventional SPPS.
Results Design, synthesis, and

testing of the DNPBS α-amino protecting group Thiols such as glutathione and cysteine are biocompatible soft nucleophiles that show striking chemical reactivity under physiological conditions. Temporary protection of bioactive molecules with thiol-liable protecting groups has been widely used in biomedical research and therapeutic applications 13 . Therefore, we reasoned that SPPS based on a thiol-liable α-amino protecting group might be superior to conventional SPPS. 2,4-Dinitrophenylsulfonyl 13 , dithiasuccinoyl 14 , and O -nitrophenylsulfenyl 15 protecting groups have been tried for this purpose, but they suffer from either difficult synthesis or inefficient removal. However, introduction and removal of the 2,4-dinitrobenzenesulfenyl group, which was developed for the protection of the 5′-OH of nucleosides, is convenient and efficient 16 . Therefore, we synthesized a series of 2,4-dinitrobenzenesulfenyl derivatives and eventually found that DNPBS was an ideal thiol-liable α-amino protecting group for SPPS. The protecting reagent, DNPBS-Cl ( 2 ), was synthesized by treatment of 1-chloro-2-phenyl-4,6-dinitrobenzene with benzyl mercaptane in the presence of Et 3 N to give 1 (94% yield), which was then treated with SO 2 Cl 2 to afford 2 (85% yield, Fig. 1b ). The amino group of benzylamine could be protected by treatment with 2 in the presence of 4-methylmorpholine to afford 3 in 90% yield (Fig. 1c ). Incubation of 3 with 1 M p -toluenethiol in pyridine at room temperature resulted in quantitative removal of the DNPBS moiety in less than 1 min (Fig. 1d ). During the deprotection step, the color of the reaction mixture changed from light yellow to dark red. Analysis of the products revealed that the released DNPBS moiety was transformed to disulfane 4 , which has a λ max of 490 nm in pyridine and thus was responsible for the red color (Fig. 1e ). In contrast, a pyridine solution of protected benzylamine (

3 ) had a λ max of 350 nm and did not absorb at 490 nm. Furthermore, neither 3 nor 4 absorbed at a wavelength of >420 nm in THF, a less polar solvent. The color change that occurs during the deprotection reaction had the advantage of allowing us to monitor the reaction visually or by UV–vis spectrometry. Preparation of DNPBS-protected amino acids Because the deprotecting reagent (1 M p -toluenethiol in pyridine) for DNPBS SPPS is weakly basic, we protected the amino acid side chains with the acid-labile protecting groups that are used for Fmoc SPPS. The side-chain-protected l -amino acids were treated with trimethylsilyl chloride and 4-methyl morpholine in dichloromethane (DCM)/acetonitrile (MeCN) and subsequently with DNPBS-Cl ( 2 ). After reaction at room temperature for 20 min followed by simple precipitation and/or recrystallization, pure DNPBS-protected amino acids 5 – 24 were obtained in 75–96% yields (Fig. 2 ). The 1 H NMR spectra of a solution of DNPBS- l -Ile-OH ( 9 ) in deuterated dimethyl formamide (DMF-d 7 ) or THF-d 8 showed no decomposition after up to 30 days at room temperature (Supplementary Fig. 1 ), indicating that DNPBS is a stable α-amino protecting group. Fig. 2: Synthesis of DNPBS-protected amino acids. a General synthetic strategy of DNPBS-protected amino acids. b Structures of DNPBS-protected amino acids. DNPBS, 2,4-dinitro-6-phenyl-benzene sulfenyl. Full size image Because some Fmoc-protected amino acids are poorly soluble in low-polarity solvents, only highly polar solvents such as DMF and N -methylpyrrolidone can be used for Fmoc SPPS 9 , 17 . In contrast, all the DNPBS-protected amino acids we prepared exhibited good solubility (>0.1 M) in a broad range of organic solvents, including but not limited to DCM, EtOAc,

Understanding OxymaPure as a Peptide Coupling Additive: A Guide to New Oxyma Derivatives. — Srinivasa Rao Manne et al., 2022

The amide/peptide bond is almost exclusive
in peptide structures,
but its presence is also the most common in organic compounds with
pharmaceutical interest as reflected in independent reports of the
Centres of Excellence for Drug Discovery (CEDD) at GlaxoSmithKline
(GSK) and of the University of Manchester. 1 , 2 Although
it looks simple, the reaction of a carboxylic acid and amine to render
the amide/peptide bond is not so straightforward and requires activation
of one of the two components. While activation of the amino function
has been increasingly studied in recent years, historically, the majority
of amide/peptide bonds considered within the pharmaceutical industry
are obtained via the activation of the carboxylic
acid group. 3
The leitmotif of this
long journey of the carboxylic group activation
is “reactivity/stability”. Thus, the activation should
be strong enough to allow amide/peptide formation but with sufficient
stability to allow the reaction before decomposition and to avoid
or minimize undesired side reactions. 3 The
pioneer studies of Fisher and Curtius exemplified this dichotomy.
While Fisher proposed the acyl chloride as the activating method, 4 Curtius developed less strong activation, the
acyl azide, 5 which was the method of choice
for peptide/amide formation until the early 1960s. Unfortunately,
neither were exempt from side reactions. 6
A real breakthrough was the development of the carbodiimide
reagents
by Sheehan, 7 which is still the most popular
coupling method. Initially, the carboxylic acid reacts with carbodiimides
and forms a reactive O -acylisourea ( 1 ) intermediate. Then, this intermediate reacts with the nucleophilic
amine and forms the corresponding amides/peptides ( Scheme 1 ). In parallel, Bodanszky introduced
the concept of active esters, 8 taking as
a model the p -nitrophenyl esters, which react smoothly
with amines giving the amide/peptide bond. With time, the use of carbodiimides
has facilitated the preparation of active esters, which could be purified,
stored for a long period of time, and even commercial

ized.
In 1970, König and Geiger proposed the use of 1-hydroxybenzotriazole
(HOBt) as an additive during carbodiimide activation. 9 HOBt reacts instantly with the O-acylisourea intermediate
rendering in situ the corresponding OBt active species.
The OBt active species, which can be found on different isoforms,
are described to be very reactive and difficult to isolate (see below).
The presence of HOBt during the mediated carbodiimide coupling translates
to better yields and less racemization of the carboxylic moiety. Although
it is commonly thought that this better performance of the carbodiimide
in the presence of HOBt is due to the higher reactivity of the OBt
active species compared to O -acylisourea ( 1 ), in fact, the opposite is true. The intermediate O -acylisourea ( 1 ) is more reactive than the OBt active
species ( 4 ). O -acylisourea ( 1 ) avoids the formation of a rearrangement side reaction that renders
the inactive N -acylurea ( 2 ) and the
formation of the oxazolone ( 3 ), which is less reactive
than the OBt active species ( 4) and, in addition, provokes
racemization ( Scheme 1 ).
For many years, the active species involved in all coupling
reactions
were OBt or OBt derivatives, mainly 6-chloro-1-hydroxybenzotriazole
(6-Cl-HOBt) and 7-aza-1-hydroxybenzotriazole (HOAt), and the related
1-oxo-2-hydroxydihydrobenzotriazine (HODhbt, HOOBt). These additives
are being used either as additives in carbodiimide-mediated coupling
or as stand-alone reagents such as N -[(1 H -benzotriazol-1-yl)(dimethylamino)-methylene]-N-methylmethanaminium
hexafluorophosphate N -oxide (HBTU), N -[6-chloro(1 H -benzotriazol-1-yl)-(dimethylamino)methylene]-

N -methylmethanaminium hexafluorophosphate N -oxide (6-Cl-HBTU, HCTU), and N -[(dimethylamino)-1 H -1,2,3-triazolo[4,5- b ]-pyridin-1-ylmethylene]- N -methylmethanaminium hexafluorophosphate N -oxide (HATU) as aminium salts; 10 − 15 and benzotriazol-1-yloxytri(pyrrolidino) phosphonium hexafluorophosphate
(PyBOP), (6-chloro-benzotriazol-1-yloxy)tris(pyrrolidino) phosphonium
hexafluorophosphate (PyClock), and (7-azabenzotriazol-1-yl)oxy]tris(pyrrolidino)
phosphonium hexafluorophosphate (PyAOP) as phosphonium salts ( Figure 1 ). 16 , 17
Some
important benzotriazole additives and benzotriazole-based
coupling reagents.
However, after September
11, 2001, the potentially explosive character
of HOBt and its triazole/triazine-related additives was reported. 18 These compounds were recategorized under a Class
1 explosive category, making their transportation difficult. 18
In this context, our groups started a
broad research project with
the goal of developing another family of safe and efficient additives,
based on a different template. Our premise for developing it was,
first, retaining the N–OH as the leaving group, because phenols
were reported in the literature to have the worst performance, and,
second, avoiding the presence of several N atoms in a row to minimize
the risk of explosion.
Our first results using N–OH heterocycles
were not very
positive, because although the additives developed were useful, their
performance was far inferior to that of 1-hydroxybenzotriazoles. Then,
we investigated the oxime series proposed by Itoh, in particular,
the ethyl 2-hydroximino-2-cyanoacetate (OxymaPure ( 1 )), 19 which

Uncovering the Most Kinetically Influential Reaction Pathway Driving the Generation of HCN from Oxyma/DIC Adduct: A Theoretical Study. — Lingfeng Gui et al., 2023

Carboxylic acid activation
is an important step in forming the
amide linkage between two amino acids, and thus has wide applications
in the synthesis of peptides and other polymers. 1 In carboxylic acid activation ( Scheme 1 ), amino acid 1 reacts with
diisopropylcarbodiimide (DIC) 2 to form a strongly activated
O-acylisourea intermediate 3 which then reacts with ethyl
(hydroxyimino)cyanoacetate (Oxyma) 4 to form an active
oxime ester 5 . Oxime ester 5 further reacts
with the amino group to form a peptide bond such that the amino acid’s
chirality is largely retained. 2 − 4 The Oxyma/DIC reagent combination
has been demonstrated to be a superior reagent combination for amino
acid activation with the merits of high coupling efficiency, inhibition
of racemization, and lower risk of explosion. 5 However, it has recently been reported 6 that Oxyma and DIC can undergo an intermolecular reaction, and generate
hydrogen cyanide (HCN) during the process of amino acid activation
in DMF at 20 °C. This can pose a serious threat to any personnel
carrying out this reaction. It has been suggested that Oxyma and DIC
first undergo an intermolecular reaction in an analogous manner to
amino acid activation by DIC to form an acyclic linear adduct 6 that can quickly decompose into a cyclic structure 7 and HCN ( Scheme 2 ). The five-membered ring structure 7 has been
assigned based on NMR spectroscopy and LC-HRMS. 6 Despite the instability of the intermediate 6 at room temperature, it has been detected with in situ NMR when
the temperature is lowered to −30 °C. The decomposition
reaction mechanism has been postulated by McFarland et al. 6 to be an intramolecular nucleophilic attack on
the oxime carbon by the sp 3 -nitrogen. However, this mechanism
has not been verified experimentally or computationally.
Erny et al. 7 attempted
to reduce
the
risk of HCN generation from the reaction of Oxyma and DIC by selecting
a different reaction solvent and scavenging produced HCN with dimethyl
trisulfide. However,

they found that HCN formation cannot be fully
suppressed in this way with continued and significant safety concerns
when the reaction is scaled up. They investigated the possible involvement
of an N-oxyl radical from Oxyma in the reaction mechanism, but this
possibility was excluded since the addition of diisopropylthiourea
(DITU) as an N-oxyl radical scavenger did not affect the production
of HCN. Manne et al. 8 found that the steric
hindrance caused by the side chains bonded to the nitrogen atoms of
the carbodiimide has a large effect on HCN formation: the carbodiimide
with two tertiary alkyl substituents, i.e., N , N ′-di- tert -butylcarbodiimide (DTBC),
does not lead to HCN formation but shows unacceptably poor performance
in peptide synthesis. The carbodiimide with two primary alkyl substituents,
i.e., N -ethyl- N ′-(3-(dimethylamino)propyl)carbodiimide
hydrochloride (EDC.HCl), forms no HCN with Oxyma but using it in peptide
synthesis is still accompanied by a reduction in the purity of the
peptide synthesized compared to DIC. The carbodiimide with the combination
of one primary alkyl substituent and one tertiary substituent, i.e., tert -butylethylcarbodiimide (TBEC), can achieve similar
or even better performance than DIC does, though currently TBEC is
more expensive and its properties need to be further investigated
before TBEC can replace DIC in the industrial manufacture of peptides. 9 In another study, Manne et al. 10 proposed a safer experimental protocol that can avoid the
production of HCN. The amino acid is preactivated with DIC for 5 min,
and the mixture is then added to peptide resin, 15 s after which Oxyma
is added. However, this protocol is also likely to increase the chance
of racemization during the process of preactivation.
To address
the safety issues caused by HCN generation without compromising
the performance of peptide synthesis, deeper insights into the reaction
mechanism and kinetics are necessary.

The objective of the current
work is to investigate systematically mechanistic and kinetic aspects
of the addition reaction of Oxyma and DIC and of the decomposition
reaction of the Oxyma/DIC adduct 6 using density functional
theory (DFT) calculations. Furthermore, a theoretical analysis is
performed to identify the rate-determining step (RDS) that best accounts
for the kinetics of the HCN generation.
All calculations are performed
using B3LYP-D3 11 /6-31+g(d) in Gaussian
16, Revision C.01 12 . Here Grimme’s
D3 dispersion 11 is used to model the London
dispersion interactions
between
species to ensure chemical accuracy. The default “ULTRAFINE”
integral grid is used. The keyword “VTIGHT” is specified.
Frequency calculations are performed at a temperature of 293 K to
compute the thermal contributions to the Gibbs free energy and confirm
that there is no imaginary frequency for the structures of reactants,
products, and intermediates, and only one imaginary frequency for
the structures of transition states. The transition state structures
are further confirmed by running Intrinsic Reaction Coordinate (IRC) 13 calculations to check whether the transition-state
structures connect the corresponding reactants and products. The SMD
continuum solvation model 14 is utilized
to simulate the solvent environment ( N , N -dimethylformamide) implicitly in geometry optimization as well as
NMR calculations. NMR spectra are computed using the Gauge-Independent
Atomic Orbital (GIAO) method 15 by specifying
the keyword “NMR” in the Gaussian input files. A conformer
search of 6 and 7 is conducted using the
GMMX add-on in Gaussview 6 with the force field MMFF94. 16 The resulting structures are then optimized
with the DFT method at the aforementioned level of theory. Throughout
the paper, Gibbs free energies are considered for the discussion.
We first investigate
the mechanism of the addition reaction of Oxyma and DIC, as it is
considered to be similar to that of an amino acid and DIC. 6 It has previously been proposed that the reaction
of an amino acid and DIC begins with a proton transfer from the carbox

Carbodiimide-Mediated Beckmann Rearrangement of Oxyma-B as a Side Reaction in Peptide Synthesis. — Andrea Orlandin et al., 2022

The rigid regulatory requirements imposed in recent years for the peptide active pharmaceutical ingredients promoted a continuous improvement of the methods for their manufacturing to obtain target compounds with high purity and yield [ 1 , 2 , 3 ]. To this aim, noticeable efforts have to be made in the development and optimization of the upstream and downstream processes. One of the most important steps of peptide synthesis is the peptide (amide) bond formation or coupling. Many efficient methods have been developed to carry out the coupling reaction with almost quantitative yield. Most of them are based on the activation of the carboxylic group of an amino acid into an electrophilic center to perform the following reaction with the amine nucleophile. This carboxylic group activation can be achieved by introducing an electron-withdrawing group at the carbonyl carbon atom, such as halide, azide, or more complex groups, which include an oxygen atom linked to a double-bonded carbon atom (O-C=), a cationic carbon (O-C + ) or phosphorus (O-P + ), or nitrogen adjacent to a double bond or double-bonded atom (O-N= and O-N-X=) [ 4 ]. However, carboxylic group activation can often facilitate a side-chain-induced racemization of sensitive amino acids, such as histidine, cysteine, serine, and threonine [ 5 ]. As a result, the manufactured peptides can contain diastereomeric impurities that are very difficult to separate without a significant decrease in yield. Various approaches have been proposed to suppress the loss of the optical purity of these susceptible amino acids during coupling reactions. For example, a careful selection of the side-chain protecting groups and a change in the solid support can noticeably decrease the amount of racemized product due to the electron-withdrawing effects and steric shielding [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ].
One of the most efficient ways to prevent racemization is the use of an additive during the activation of the amino acid, such as 1-hydroxybenzotriazole (HOBt) or OxymaPure [ 13 , 14 ]. Despite the broad range of applicability, these additives show limited ability to suppress the racemization of susceptible amino acids [ 5 ]. In this regard, Jad et al. recently proposed the novel reagent Oxyma-B

(1,3-dimethylvioluric acid) as an additive for the peptide coupling reaction ( Figure 1 ) [ 15 , 16 , 17 , 18 ].
Oxyma-B can be easily prepared from 1,3-dimethylbarbituric acid by reaction with sodium nitrite in the presence of potassium hydroxide and acetic acid, and it affords more efficient control of the optical purity during the coupling reaction than other OxymaPure- and benzotriazole-based reagents. For example, Oxyma-B in combination with diisopropylcarbodiimide (DIC) was a more potent racemization suppressor than OxymaPure/DIC for His coupling in the synthesis of the H-Gly-His-Phe-NH 2 tripeptide (1% vs. 3% of D-His isomer) [ 15 ].
Both OxymaPure and Oxyma-B belong to the class of oximes, which are known to be highly reactive compounds and can trigger various side reactions, such as Beckmann rearrangement to form substituted amides in the presence of strong acids and other activators of the oxime hydroxyl group [ 19 , 20 , 21 ]. To test the efficiency of Oxyma-B for industrial applications, we used this reagent for the preparation of various His-containing peptides. Surprisingly, in analyzing the high-performance liquid chromatography (HPLC) profiles of the isolated peptides, we found two impurities with the same molecular weight and overall content of up to 15%. The formation of these impurities can drastically decrease the yield of the target peptides, especially when multiple cycles of coupling with this additive have to be carried out. Accordingly, to understand the origin of this behavior not yet reported and to find the best reaction conditions to prevent the formation of these impurities, we synthesized them and studied their structures in detail.
Histidine is present as the N -terminal amino acid of several commercially relevant peptides, such as Glucagon and GLP-1 analogs, which comprise the blockbuster drugs Liraglutide and Semaglutide currently used for diabetes treatment [ 22 , 23 ]. These peptides have a strong tendency to aggregate and to generate several side reactions during conventional step-by-step solid-phase synthesis, which noticeably complicates their preparation, particularly when the last residues have to be coupled

[ 24 ]. To solve this problem, the use of a fragment condensation approach was selected as an excellent alternative, where protected peptide fragments are prepared separately and assembled to obtain the complete peptide sequence [ 25 , 26 ]. Accordingly, we carried out the synthesis of the N -terminal Glucagon fragment Boc-His(Trt)-Ser( t Bu)-Gln(Trt)-Gly-OH (abbreviated as HSQG later in the text), using DIC as a coupling reagent and Oxyma-B as an additive for the last coupling involving the His residue. As expected, Oxyma-B efficiently prevented the epimerization of histidine, resulting in only 0.09% of the D-His-diastereomer in the isolated HSQG sequence ( Figure 2 A).
However, the HPLC profile of the isolated crude HSQG peptide showed the presence of two peaks of unexpected impurities with an overall area of 7.4% (with respect to the target product peak). The corresponding m/z value of these two impurities showed the same molecular weight, corresponding to the absence of the N -terminal histidine and the condensation of one molecule of Oxyma-B to the tripeptide H-Ser( t Bu)-Gln(Trt)-Gly-OH (with the concurrent elimination of one molecule of water). Furthermore, a gradual conversion of one HPLC peak to another one was observed over time, indicating that one of them could be an unstable intermediate. Lastly, the HPLC chromatogram of the crude Glucagon, which was prepared by on-resin condensation of fragment 5–29 with HSQG followed by the cleavage from the solid support, showed the presence of an equivalent amount (about 7.5%) of des-His-Glucagon, confirming the incomplete coupling of the His residue ( Figure 2 B).
To find an explanation for this unexpected result, we investigated the reaction using a series of model experiments with different amines and amino acids ( Figure 3 ).
Several amino acids linked to 2-chlorotrityl chloride resin (CTC) were treated with the mixture of Oxyma-B and DIC in dimethylformamide. Oxyma-B-derivatives corresponding to the impurity observed in the synthesis of HSQG were found in the case of the α-amino acids with a

Dipropylamine for 9-Fluorenylmethyloxycarbonyl (Fmoc) Deprotection with Reduced Aspartimide Formation in Solid-Phase Peptide Synthesis. — Hippolyte Personne et al., 2023

Solid-phase peptide synthesis (SPPS) with
fluorenylmethyloxycarbonyl
(Fmoc) as the α-amino protecting group for amino acid building
blocks is currently the dominant synthesis method for peptide research
and manufacturing. The Fmoc protecting group is removed by a base,
which triggers β-elimination of carbamic acid followed by the
formation of an adduct with the dibenzofulvene (DBF) byproduct ( Figure 1 a) with a nucleophile. 1 Piperidine (PPR) is currently the most widely
used Fmoc removal reagent. However, in addition to its toxicity and
regulation, PPR induces the formation of aspartimide in some aspartic
acid-containing sequences, which can hydrolyze to α- or β-peptides,
react again with the nucleophile to form peptide-base derivatives,
or induce an intramolecular formation of the terminating diketopiperazine
byproduct by nucleophilic attack of the deprotected amino group of
the next amino acid ( Figure 1 b,c). 2 − 6
(a)
Mechanism of Fmoc deprotection and trapping of dibenzofulvene.
(b) Mechanism of aspartimide formation, its hydrolysis to α-
or β-peptides, and its ring-opening by nucleophilic attack to
α- or β-peptide-nucleophile adducts. (c) Mechanism of
diketopiperazine byproduct formation. (d) Structural formulae of reagents
used for Fmoc removal.
PPR can be replaced by a mixture of piperazine
(PZ) as the nucleophile
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the base 7 or simply DBU without added nucleophile ( Figure 1 d); 8 however, DBU is quite expensive and produces a considerable
amount of aspartimide for aspartimide-prone sequences. One can also
add weak acids such as formic acid or ethyl cyanohydroxyiminoacetate
(Oxyma) to temper the basicity of the PPR solution to reduce aspartim

ide
formation. 9 However, this still does not
solve the cost, stench, and availability issues of PPR.
Alternative
bases 10 − 13 or aspartate side-chain protecting groups 14 − 17 have been reported to overcome
the limitations of PPR or PZ/DBU; however, none of them combines low
cost and convenient use with low aspartimide and good yields. Here,
we searched for PPR alternatives in the context of a high-temperature
(60 °C) SPPS protocol with Oxyma and N , N ′-diisopropylcarbodiimide (DIC) as coupling reagents 18 and N , N -dimethylformamide
(DMF) as the solvent, which in our hands work excellently for a variety
of peptides, cyclic peptides, and peptide dendrimers. 19 − 22 We noted that diethylamine (DEA, b.p. 55 °C) has been used
for Fmoc removal in process-scale SPPS. 23 We therefore set out to test the less-volatile dipropylamine (DPA,
b.p. 110 °C), which is advantageously cheaper than both DEA and
dibutylamine (DBA).
Due to the lower basicity
of DPA (p K a = 10.9) compared to PPR (p K a = 11.1), we investigated whether DPA might
solve the issue of aspartimide formation in SPPS of aspartimide-prone
sequences using the prototypical test case hexapeptide 1 (VKDGYI) and compared it to other Fmoc deprotecting reagents.
Aspartimide formation is catalyzed by relatively strong bases, and
lowering the basicity allows one to reduce the formation of this side
product. For instance, the crude product of hexapeptide 1 synthesized using PPR for Fmoc removal contained 17% aspartimide.
The results were even worse with DBU, which is a stronger base than
PPR. In this case, purity was only 52% due to 25% aspartimide and
23% byproducts. Furthermore, using PZ/DBU only gave byproducts ( Table 1 ).
PPR, piperidine

; PZ, piperazine;
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DPA, dipropylamine; DEA,
diethylamine; DBA, dibutylamine. Percentages (%) are in w/v in the
case of PZ and in v/v otherwise.
Crude yield is calculated as follows:
(crude mass/molecular weight of desired peptide)/(mass of resin ×
resin loading) × % of desired product content in crude.
Product ratio was determined by
LC analysis and is given as follows: % desired product/% aspartimide/%
other byproducts. The main byproduct observed was the diketopiperazine
terminating sequence (mass 576.3 Da); see HRMS data in the Supporting Information .
By contrast, the crude product of hexapeptide 1 synthesized
using DPA for Fmoc removal was 96% pure and contained only 4% aspartimide
as the only detectable byproduct. We obtained similar SPPS yields
with hexapeptide 1 using the secondary aliphatic amines
DEA and DBA for Fmoc removal, although some byproducts were also observed,
whereas sterically hindered diisobutylamine (DIBA) only gave byproducts
( Table S1 ). Furthermore, we did not detect
any trace of 1β (VKD(β)GYI), the β-peptide
analogue of hexapeptide 1 , which can potentially be formed
by reopening of the aspartimide, upon 1 H NMR analysis in
comparison with an independently synthesized β-peptide sample
(Supporting Information Figures S1 and S2 ).
Note that aspartimide formation was strongly reduced by
adding
0.5 M Oxyma or hydroxybenzotriazole (HOBt) as weak acids to PPR ( Tables 1 and S1 ), reproducing published results. 9 Adding Oxyma also allowed one to obtain the product
with PZ/DBU; however, adding Oxyma to DPA did not reduce aspartimide
formation further compared to DPA alone. When tested at 90 °C,
SPPS of hexape

Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine? — Omar F. Luna et al., 2016

1. IntroductionThe 9-fluorenylmethoxycarbonyl (Fmoc) strategy is the most used strategy in solid phase peptide synthesis (SPPS) and remains valid even forty years after its implementation [1], thanks to the constant development and improvement in reagents and strategies for the different steps [2,3,4,5].Although it is commonly understood that coupling is the most demanding reaction in the whole synthetic process, the α-amino deprotection step is also crucial in order to secure the quality of the target peptide. Poor efficiency in deprotection will result in decreased yield and quality due to deleted residues and even capped peptides, which also generates the need of additional purification steps [1,6]. Contrary to tert-butoxycarbonyl (Boc) chemistry, where the α-amino deprotection is carried out in trifluoroacetic acid (TFA), which is the best solvent/reagent to disaggregate the peptide chain, Fmoc removal is carried out in N,N-dimethylformamide (DMF), which is a worse solvent to disrupt the interchain aggregation, very often favored by the presence of the own Fmoc group [6].Fmoc group removal in solid phase peptide synthesis (SPPS) proceeds through a two-step mechanism: the removal of the acidic proton at the 9-position of the fluorene ring system by a mild base, preferably a secondary amine, and the subsequent β-elimination that yields a highly reactive dibenzofulvene (DBF) intermediate which is immediately trapped by the secondary amine to form stable adducts (Scheme 1). These reactions work better in an electron donor and relative polar medium (DMF or N-methylpirrolidone [NMP]) compared to a relatively non-polar one (dichloromethane [DCM]) [6,7].Although, the Fmoc group can be easily removed by primary amines and, less easily, by tertiary amines, the most convenient method involves the use of cyclic secondary amines due to their nucleophilicity. These amines trap very efficiently the DBF intermediate generating an adduct and, therefore, driving the deprotection step to completion [8,9,10,11]. The most used secondary amine is piperidine (

pKa = 11.1, 25 °C), although one of the main reported problems with its use is the formation of aspartimide [12,13], which can be minimized by the use of other bases.Additionally, availability is its major drawback; in fact, piperidine has a current legal status as a controlled substance regulated by the Drug Enforcement Agency (International Narcotics Control Board for 2014), because it may be used as a precursor of illegal psychotropic drugs. This fact implies that special permission is required for purchasing piperidine which, in certain institutions or countries, can cause administrative problems. In this regard, other cyclic secondary amines, 4-methyl piperidine, or piperazine (pKa = 10.78 and 9.73, 25 °C respectively) have been also proposed [8,9,10].This report presents the comparison of three strategies for Fmoc removal using microwave-assisted peptide synthesis to investigate if the use of 4-methylpiperidine (4MP) or piperazine (PZ) is comparable in terms of efficiency with the use of piperidine (PP) and, therefore, can replace it. To this respect, this study has been carried out using four sequences in high-demand production in our laboratory, of medium-large size peptides (up to 26 residues), which allows a realistic context for making the comparison of the deprotection conditions (Table 1), using a Rink amide resin, and a Liberty Blue™ microwave automated synthesizer. Peptides were characterized by high-performance liquid chromatography (HPLC), mass spectrometry and circular dichroism in order to determine purity, presence of byproducts, and the secondary structure of the resulting peptides. In addition, deprotection kinetic assays were conducted using arginine and leucine as initial amino acids to complement and corroborate our results. 2. Results 2.1. Yield and Purity of Synthesized PeptidesTotal crude yield, purity, and peptide-specific yield (as defined in Materials and Methods) were obtained according to the HPLC data analysis of the crude and purified product (Supplementary Materials Tables S1–S4 and Figures S1–S4). These results are summarized in Table 2.Each peptide showed similar results, regardless of the deprotection reagent used. Table 2 summarizes these results.Each peptide showed nearly the same

values in total crude yield, purity, and peptide-specific yield with the three deprotection reagents. Peptide NBC1951 showed the greater variation among the piperidine and the other two deprotection reagents. PP showed the best results in purity and peptide-specific yield (the two are related), except in the case of peptide NBC112 where better yields (crude yield and peptide-specific yield) were obtained with 4MP, but better purity was obtained with PZ. 2.2. HPLC and Mass SpectrometryResults of HPLC and mass spectrometry (electrospray ionization (ESI)-MS) for crude synthetic peptides are summarized in Figure 1 and Table 3.As observed, the chromatograms and mass spectra for the three deprotection strategies are superimposable with regard to the main components; however, in each specific case there are differences in the number of species obtained in the crude product (Tables S1–S4).The enrichment of the peptide through the C18 extraction columns with an acetonitrile:water gradient only allowed purification in the case of the peptide NBC155; with the other peptides enrichment occurred, but the product was accompanied by other substances (Figures S1–S4).Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis of the specific peaks in HPLC of enriched products showed specific sequence deletions, which can come from either incomplete incorporation of the deleted residues or incomplete removal of the Fmoc from the previous residue (Table 4):
(1)NBC112: Alanine and histidine deletion with 4MP and PZ with a %area higher than 10%.(2)NBC759: Lysine deletion with a %area higher than 10% in all cases.(3)NBC1951: Glutamine/lysine deletion with a %area higher than 10% in all cases, the two amino acids co-eluted in the chromatogram (Figure S4 peak 1). 2.3. Secondary Structure CharacterizationCircular dichroism showed an alpha helix tendency for all four peptides (Figure 2), in accordance with previous reports for NBC155 and NBC1951 [16,19]. Pepfold3 server [20] prediction for the secondary structure also showed the alpha helix as the best model for

Morpholine, a strong contender for Fmoc removal in solid-phase peptide synthesis — Sinenhlanhla N. Mthembu et al., 2023

1 INTRODUCTION

     The increasing number of peptide-based therapeutics approved by the Food and Drug Administration (FDA) each year has brought about a boom in peptide pharmaceuticals and the related manufacturing sector.1, 2 Like the manufacturing process of any other pharmaceutical, that of these peptidyl drugs requires optimization, good manufacturing practices, and overall sustainability to meet the requirements of regulatory authorities.3, 4 In this regard, countless initiatives and efforts are underway to cover the growing demand for the development of sustainable chemical syntheses for these compounds.5-7 Generally, solution-phase synthesis, solid-phase synthesis, and/or a combination of the two have been adopted for peptide production.8, 9 In this context, 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (SPPS) is popular among chemists, mostly because of the mild conditions used.10, 11 Successful peptide synthesis using Fmoc-SPPS protocols involves the efficient execution of two repetitive steps, namely, the incorporation of Fmoc-protected amino acids onto a growing peptide chain attached to a solid support and the removal of Fmoc using base after each incorporation.12

     Fmoc is a temporary protecting group for Nα-amino acids, and it can be rapidly removed by treatments with a variety of primary or secondary amines.10, 13 Generally, excess base in a relatively polar solvent can remove Fmoc efficiently.10 In this regard, cyclic secondary amines are the bases of choice in Fmoc-SPPS because of the advantage of the scavenging dibenzofulvene generated during Fmoc removal. Figure 1 shows some of these widely used secondary amines arranged on the basis of increasing pKa values.14-16




              FIGURE 1Open in figure viewerPowerPoint



              Cyclic secondary amines used for Fmoc group removal and their corresponding pKa values.




     The use of these secondary amines renders successful Fmoc-SPPS protocols, although a plethora of side reactions occur.17 These reactions include the formation of diketopiperazine (DKP), epimerization, and formation of aspartimides and related side products, to name just a few. For piperidine (PIP), the most popular base used to remove Fmoc, the extent of these side reactions often implies a greater challenge in downstream processing, including a loss in the yield of the desired peptide. PIP is a controlled substance in terms of narcotic drug production and as such its use is restricted. Moreover, and most importantly, it is a hazardous chemical with a greenness score of 6.9 on a scale of 0–10 in GSK's reagent selection guide.18 Also, for greener alternatives to PIP, such as 4-methylpiperidine (4MP), there have been fewer reports of improvements concerning the minimization of base-induced side reactions.19-21 In this regard, we recently reported the use of 30% 4MP in 0.5 M OxymaPure–DMF for Fmoc removal.22 In this context, other secondary amines, such as pyrrolidine and piperazine (PPZ), have also been exploited on several occasions. For example, the use of PPZ supplemented with DBU in N-methylpyrrolidone (NMP) has been described to enhance the suppression of DKP formation in some sequences.23 However, several factors, including limited solubility in DMF, reduced base strength and thus necessitating the addition of another stronger base, and the requirement of additives like hydroxybenzotriazole (HOBt)24 or formic acid25 to suppress side reactions, circumscribe the general use of PPZ in Fmoc-SPPS. Likewise,

the performance of pyrrolidine was not promising due to its higher base strength, similar to that of PIP, thus influencing side reactions and sometimes causing the degradation of the desired peptide.26, 27 In contrast, in the case of morpholine (Morph), Fmoc removal is generally accompanied by fewer side reactions. However, the use of Morph requires longer reaction time when compared to other cyclic amines.28-30 Owing to its lower base strength, to date, the use of Morph has been limited to a couple of Fmoc removal cycles for peptide sequences prone to side reactions during SPPS.31 For instance, 20% Morph-NMP was used for Fmoc removal from the Asp-X (X = His or Ser) motif in sequences to suppress aspartimide formation.32, 33 There are also reports of Fmoc removal using 50% Morph–DMF to suppress base-catalyzed side reactions.34-37

     Here we studied the performance of Morph as a general alternative to PIP for Fmoc removal in SPPS and evaluated its capacity to minimize base-catalyzed side reactions, namely, DKP and aspartimide formation, and epimerization and β elimination when Cys is the C-terminal amino acid in the synthesis of peptide acids. To this end, we first tested the capacity of Morph to remove Fmoc by varying the proportion of Morph–DMF over different time intervals. With the best condition obtained through optimization, several experiments were then carried out to study the severity of prominent base-influenced side reactions in SPPS. The optimized Morph–DMF deprotection condition was tested for both DKP and aspartimide formation. Finally, the use of Morph–DMF as an Fmoc removal reagent was validated during the synthesis of linear somatostatin as a model peptide.

2 EXPERIMENTAL SECTION

        2.1 Materials and methods

        F

Computer vision as a new paradigm for monitoring of solution and solid phase peptide synthesis. — Chunhui Yan et al., 2023

Amides are one of the most frequently occurring functional groups in pharmaceutically-relevant chemistry, requiring reliable synthetic transformations to make them. 1–3 Their preparation generally requires activation of the carboxylic acid to promote a condensation reaction ( Fig. 1 ). 4 The acid is typically activated in the form of an acyl chloride ( Fig. 1A ), acyl azide ( Fig. 1B ), anhydride ( Fig. 1C and D ), or active ester ( Fig. 1E ). Despite presenting one of the simplest methods of activation, 1 the acyl chloride approach comes with a major drawback in the release of HCl, limiting applications involving acid-labile substrates. 4 At elevated temperatures, using acyl azides can result in peptide urea byproducts (Curtius rearrangement, Fig. 1B ), which are difficult to separate from the desired product. 5 When using symmetric anhydrides ( Fig. 1C ), only half of the parent acid is coupled, wasting the other half, while regioselectivity between the two electrophilic positions of a mixed anhydride ( Fig. 1D ) can lead to inefficiencies. While these methods still see use in industry, these drawbacks have directed our focus towards amidations mediated by coupling reagents. Specifically, we explored new amide reaction monitoring methods using 1-hydroxy-7-azabenzotriazole (HOAt)-based coupling reagents, as these are arguably the most important class of available amide bond forming methods ( Fig. 1E ).
Amidation reactions involving coupling reagents 6 often produce distinct colour changes as a result of the anionic by-product. 7,8 In our case, coupling reagents based on HOAt ( Fig. 2 ) 9 change from colourless to yellow, as the reaction progresses. A range of aminium/uronium or phosphonium reagents have since been developed and become widely available, to improve stability and ease of handling. 5,6,10 Fig. 2 presents the proposed mechanism for amide coupling reactions mediated by HOAt-based reagents, using HATU as an example. The acid substrate is activated as the OAt ester which then reacts with the amine nucleophile. The HOAt anion released during the reaction is yellow, providing a colorimetric indicator of reaction progress. To the best of our knowledge, related ac

ylation phenomena have only been monitored by Sheppard, whose team fashioned a photometer to monitor resin yellowing in a column reactor. 11 More broadly, UV-vis spectroscopic methods have been applied to solid phase peptide synthesis. 12–15 However, to date, these colour changes have not been widely used for online quantitative monitoring of amide and peptide synthesis using readily-accessible camera technology.
The incentive to investigate new approaches to monitoring amide formation was driven by the need for more direct, adaptable, digital-ready, non-contact methods of monitoring SPPS. There are two main reactions in the SPPS cycle – Fmoc deprotection and amide coupling. Both can be monitored by halting the reaction and taking samples for off-line analysis. The quality of the intermediate peptide (anchored to the solid phase resin) can be checked, either by HPLC after acidic cleavage of the peptide from the resin, or directly on resin by IR. 16 The Kaiser test, 17 and modern variations thereon, 18 are often used to provide a colorimetric test for the present of unreacted amine after an SPPS step has concluded. These monitoring methods require the user to pause the synthesis process, making turnover time slow and difficult to automate, during reaction optimisation. Thus, several methods have been reported to provide real-time monitoring based on the electrochemical property changes, 19 or physical properties, such as pressure, 20 UV absorbance, 21 and refractive index. 22 During the revision of this manuscript, Wang and co-workers released a study demonstrating the value of NMR kinetics in elucidating mechanistic aspects of amide coupling. 23 For monitoring of liquid phase peptide synthesis, Livingston's team have reported a powerful advance in UHPLC-MS. 24 Gómez-Bombarelli and Pentelute have demonstrated the value of UV-vis analysis of in-flow Fmoc deprotection reactions to generate data-rich input to build predictive deep learning insights for Fmoc deprotection efficiency. 15,25 Otake and co-workers have demonstrated the value of in-line near infrared (NIR) flow cells as a means of tracking liquid phase peptide synthesis in flow. 26
We used our recently-developed imaging kinetics software, Kineticolor , 27,28 to provide camera-enabled ex situ (non-contact) reaction monitoring ( Fig. 3 ). Looking

towards complementing (not replacing) known reaction monitoring methods used in amide and peptide synthesis, we sought to demonstrate the ability to derive useful kinetic information from the visible bulk of the reactor, and not the molecular specifics typically captured by established in situ or offline techniques. 29 In this study, we focus on the development of kinetic computer vision methods to support reaction monitoring in both liquid and solid phase peptide synthesis.
Computer vision for analytical chemistry (CVAC) 30,31 is frequently used through single image analysis techniques, providing a promising means of non-invasive analysis using digital camera technology. Comparatively, the extraction of time-dependent colourimetric information from videos of chemical reactions is less mature. 32–39 In a previous study, our computer vison-enabled reaction monitoring method helped develop the understanding of palladium catalyzed borylation and catalyst degradation phenomena from colourimetric time profiles of the reaction bulk. 28 The same methods have since been extended to tracking the impact of reaction scale-up on mixing times for process chemistry applications. 27 Considering the colour change evident in amide coupling reactions, we aimed to expand the application domain of non-contact video analysis to the widely-used amidations described above, for both solution and solid phase reaction environments.
The first amide coupling reactions to be monitored by video and analysed with the Kineticolor software focused on HATU, one of the most commonly used amide coupling reagents. These reactions formed our initial proof-of-concept. 4,6,40 In order to enable off-line HPLC analysis, we employed a sterically encumbered amine to slow the rate of amide bond formation, and thus the release of coloured OAt anion. Under the conditions shown in Fig. 4 , HPLC analysis captured a gradual increase in the amide product over 3 hours. A colourless to translucent yellow colour change was observed. As described in our earlier reports, the video recording was analysed using a range of colour models 27,28 Delta E (or Δ E ), a measure of colour change, was derived from the CIE-Lab* colour space ( Fig. 3 ), and calculated over time. In this calculation, each video frame was referenced against the first. Therefore the calculated Δ E value compares the analysed frame's colour to the colour of the first frame. As hypothesised, this approach enabled ex situ capture of the colourless to yellow colour change, registering a Δ