Revisiting NO2 As Protecting Group of Arginine in Solid-Phase Peptide Synthesis

International Journal of Molecular Sciences

Article Revisiting NO2 as Protecting Group of Arginine in Solid-Phase Peptide Synthesis

1, 1,2, 3 1,4,5,6, Mahama Alhassan † , Ashish Kumar †, John Lopez , Fernando Albericio * and Beatriz G. de la Torre 2,*

1 Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa; [email protected] (M.A.); [email protected] (A.K.) 2 KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4041, South Africa 3 Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel, Switzerland; [email protected] 4 CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, 08028 Barcelona, Spain 5 Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain 6 Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain * Correspondence: [email protected] (F.A.); [email protected] (B.G.d.l.T.) Both authors contributed equally. † Received: 27 May 2020; Accepted: 17 June 2020; Published: 23 June 2020

Abstract: The protection of side-chain arginine in solid-phase peptide synthesis requires attention since current protecting groups have several drawbacks. Herein, the NO2 group, which is scarcely used, has been revisited. This work shows that it prevents the formation of δ-lactam, the most severe side-reaction during the incorporation of Arg. Moreover, it is stable in solution for long periods and can be removed in an easy-to-understand manner. Thus, this protecting group can be removed while the protected peptide is still anchored to the resin, with SnCl2 as reducing agent in mild acid conditions using 2-MeTHF as solvent at 55 ◦C. Furthermore, we demonstrate that sonochemistry can facilitate the removal of NO2 from multiple Arg-containing peptides.

Keywords: δ-lactam formation; microwave; NBP; orthogonal protection; protecting group; sonochemistry; side-reaction; solid-phase peptide synthesis; ultrasounds

1. Introduction Arginine (Arg) is a key natural amino acid that is found in many biologically active peptides [1]. Its characteristic structural feature is the presence of a guanidino group in its side-chain. This group is strongly basic, with a pKa value of 12.5, which means the side-chain remains protonated under physiological conditions [2,3]. This guanidino moiety is key for the biological activity of Arg-containing peptides [4]. In this regard, several active pharmaceutical ingredients (APIs) contain one or even several Arg residues. For instance, this intriguing amino acid is found in desmopressin, leuprolide, and the more recently approved abaloparatide, angiotensin II, bremelanotide and afamelanotide from the α-melanocyte-stimulating hormone (α-MSH), semaglutide and other drugs approved earlier from the glucagon-like peptide family (GLP) [5]. It is important to highlight the case of etelcalcetide, which was approved by the FDA in 2017 for the treatment of secondary hyperparathyroidism in adults with chronic kidney disease on hemodialysis. It is a simple octapeptide formed by a linear chain of seven D-amino acids (one D-Cys, one D-Ala, and ﬁve D-Arg) linked to an L-Cys through a disulphide bridge [6,7]. Multiple Arg residues are also very common in antimicrobial peptides (AMPs) and the so-called cell-penetrating peptides (CPPs) [8–10]. In fact, the unique characteristic of most CPPs is

Int. J. Mol. Sci. 2020, 21, 4464; doi:10.3390/ijms21124464 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4464 2 of 12 the presence of several Arg residues, as exemplified in the fragment 48–60 of the transactivator of transcription (TAT) of HIV and octa-Arg [11]. The majority of peptides used for research and industrial purposes are prepared by a chemical process, the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) Solid-Phase Peptide Synthesis (SPPS) approach being the most convenient [12]. Although the protonated guanidino group of Arg should show poor reactivity in front of acylation, which is the key reaction in peptide synthesis, it has to be protected to facilitate its solubility in the common solvents used in SPPS and, more importantly, to avoid the two possible side-reactions. On one hand, some Orn can be formed during the stepwise peptide chain elongation or during the cleavage [13,14], and on the other hand, the most important side-reaction, the δ-lactam formation, is a process driven by the six-member ring formed [15]. δ-Lactam formation takes place during the coupling step, when the carboxylic group of Arg is activated. In contrast to other side-reactions, this cyclic amide is not directly converted into an impurity in the target peptide. Instead, it causes an impractical consumption of the activated Arg, which in turn can be indirectly translated into incomplete incorporation of the Arg and therefore the presence of a deletion (des-Arg) peptide. To mitigate this, repetitive couplings of protected Arg are carried out, a protocol that increases the cost of the whole synthetic process. This drawback is exacerbated during the industrial preparation of Arg-containing peptides, where minimum excesses of reagents are used and repetitive couplings imply high solvent and reagent consumption and a longer process time, and therefore a greater overall production cost. Of note, in an industrial mode, the most currently used Arg derivative (Fmoc-Arg(Pbf)-OH, Pbf for 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, see below) is the most expensive of all protected proteinogenic amino acids. Thus, in a 100-g scale, the cost of Fmoc-Arg(Pbf)-OH is approximately 10 times more than Fmoc-Phe-OH (€360 vs. €36). Furthermore, it is important to consider atom economy, which is 0.27 for the former and 0.40 for the latter. These figures mean that the synthesis of a peptide containing Arg is approximately 15 times more expensive in comparison with those containing Phe or other amino acids. In this scenario, approaches to optimize Arg protection, coupling, and protecting group removal could have an important impact in the industrial sector. The protection of Arg in the Fmoc strategy has its roots in tert-butoxycarbonyl (Boc) chemistry, where Arg is protected with the tosyl (Tos) group, which is removed by anhydrous HF, trifluoromethanesulfonic (TFMSA) or related acids [16]. Thus, Yajima’s group developed mesityl-2-sulfonyl (Mts) [17], and later Masahiko et al., after the in-depth study of several candidates, developed the more labile 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr) [18], which is removed with almost neat trifluoroacetic acid (TFA) in the presence of scavengers over a prolonged period. An important breakthrough in this field was the development of the 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (Pmc) by Ramage and co-workers [19,20]. The structure of this group recasts the methoxy in position 4 on Mtr in a cyclic ether and keeps three methyl groups on the aryl moiety. Pmc is more labile than Mtr. Carpino and co-workers went a step further when they discovered that the five-member ring present in the Pbf makes this group more labile than the six-member ring of the Pmc [21]. Verdini et al. [22] developed the bis-Boc protection, blocking both ω ω’ N and N for the guanidino group of Arg. Both Boc groups are removed by TFA-H2O (95:5) at room temperature (rt) in 1 h. Although our group has reported that 1,2-dimethylindole-3-sulfonyl (MIS) is much more labile than the Pbf group [23], the latter continues to be the most widely used protecting group for the side-chain of Arg. However, in addition to its high price, which is mainly due to an extremely difficult production process, Pbf is not exempt from the side-reactions outlined earlier, such as δ-lactam formation as has been recently reported by our group [24] (see Figure1 for the structures of these protecting groups). Int. J. Mol. Sci. 2020, 21, 4464 3 of 12 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 12

Figure 1. MostMost used used Arg Arg side-chain protecting groups.

In the context of SPPS, little attention has been paid to the use of the NO2 group, which was In the context of SPPS, little attention has been paid to the use of the NO2 group, which was introduced by Bergmann et al. [25] at the beginning of the era of protecting groups and has been applied introduced by Bergmann et al. [25] at the beginning of the era of protecting groups and has been mostly in solution chemistry. The strong electron-withdrawing character of the NO2 group reduces applied mostly in solution chemistry. The strong electron-withdrawing character of the NO2 group the basic nature of the guanidino group, thereby modulating its reactivity. Catalytic hydrogenation reduces the basic nature of the guanidino group, thereby modulating its reactivity. Catalytic using a wide range of catalysts such Pd black, [26] SnCl2,[27] and TiCl3 [28] has been proposed for hydrogenation using a wide range of catalysts such Pd black, [26] SnCl2, [27] and TiCl3 [28] has been the removal of the NO2 group. Catalytic hydrogenation is only friendly used in organic chemistry proposed for the removal of the NO2 group. Catalytic hydrogenation is only friendly used in organic laboratories, but not in those devoted to other biosciences. Furthermore, this reaction works relatively chemistry laboratories, but not in those devoted to other biosciences. Furthermore, this reaction without difficulty for shorter peptides containing a single Arg(NO2) residue. However, with multiple works relatively without difficulty for shorter peptides containing a single Arg(NO2) residue. Arg(NO2) residues, catalytic hydrogenation can lead to difficulties, which include a long reaction time However, with multiple Arg(NO2) residues, catalytic hydrogenation can lead to difficulties, which withinclude the a concomitant long reaction degradation time with the of the concomitant target peptide degradation [29]. Conversion of the target of a Arg(NOpeptide 2[29].) residue Conversion into an aminoguanidino-derivative [30] and reduction of the aromatic ring of a Trp and even Phe residue [31] of a Arg(NO2) residue into an aminoguanidino-derivative [30] and reduction of the aromatic ring of ahave Trp beenand even observed Phe residue in some [31] cases. have been observed in some cases. Here we revisit the use of the NO2 group for Arg protection in SPPS. In this regard, we Here we revisit the use of the NO2 group for Arg protection in SPPS. In this regard, we first first compared the stability of the NO2 derivative of Arg with the Pbf and (Boc)2 derivatives in compared the stability of the NO2 derivative of Arg with the Pbf and (Boc)2 derivatives in N,N- N,N- N- dimethylformamidedimethylformamide (DMF) (DMF) and andthe thegreen green solvent solvent N-butylpyrrolidonebutylpyrrolidone (NBP), (NBP), as as well well as as their δ tendency toto render render -lactamδ-lactam formation formation in both in both solvents solvents [32]. We[32]. then We developed then developed a method a formethod removing for the NO2 group without the concourse of catalytic hydrogenation. removing the NO2 group without the concourse of catalytic hydrogenation. 2. Results and Discussion 2. Results and Discussion 2.1. Stability of Fmoc-Arg(x)-OH in Solution 2.1. Stability of Fmoc-Arg(x)-OH in Solution The stability of three Fmoc-Arg(X)-OH analogues [X = (Boc)2, NO2, and Pbf] was studied in solutionThe overstability a period of three of time. Fmoc-Arg(X)-OH Solutions (0.2 M) analogues of each analogue [X = (Boc) in both2, NO DMF2, and and Pbf] NBP was were studied prepared in solutionin closed over high-performance a period of time. liquid Solutions chromatography (0.2 M) of (HPLC) each analogue vials following in both the DMF procedure and NBP described were preparedpreviously in by closed our group high-performance [24]. The reaction liquid was chro monitoredmatography by reverse(HPLC) phase vials following HPLC (Figures the procedure S1–S6). described previously by our group [24]. The reaction was monitored by reverse phase HPLC (Figure Fmoc-Arg(Boc)2-OH slowly degraded in both solvents over time, rendering mainly the mono S1–S6). protected Fmoc-Arg(Boc)2-OH, which was already present in the commercial sample (Table1). Fmoc-Arg(Boc)2-OH slowly degraded in both solvents over time, rendering mainly the mono Nevertheless, solutions of Fmoc-Arg(Boc)2-OH in the two solvents could be kept up to one week, protectedthereby making Fmoc-Arg(Boc) it compatible2-OH, with which peptide was synthesizers already present and also in forthe industrialcommercial manufacturing sample (Table where 1). Nevertheless, solutions of Fmoc-Arg(Boc)2-OH in the two solvents could be kept up to one week, thereby making it compatible with peptide synthesizers and also for industrial manufacturing where

Int. J. Mol. Sci. 2020, 21, 4464 4 of 12 the solution is prepared at the time of coupling. For longer periods, the degradation was slightly Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 12 higher in NBP. In contrast, the NO2 and Pbf analogues were totally stable.

theTable solution 1. Stability is prepared of Fmoc-Arg(X)-OH at the time inof DMFcoupling. (N,N- dimethylformamide)For longer periods, and the NBP degradation (N-butylpyrrolidone) was slightly higherat room in NBP. temperature. In contrast, the NO2 and Pbf analogues were totally stable. Stability was examined again at 45 °C in DMF and NBP, in the presence of OxymaPure, which are conditions commonly used in0 SPPS. h Again, 1 h Pbf 24 and h NO 482 analogues h 10 dshowed 15 total d stability 20 d (Figure 30 d

S7-S10),Fmoc-Arg(Boc) while the2-OH, bis DMFBoc derivative88.8 degraded 88.6 slight 86.9ly faster 85.0 than 77.6in the previous 65.1 experiment, 58.5 51.2 but could still be considered compatible with the standard reaction times for coupling (up to 4 h). As in Fmoc-Arg(Boc)2-OH, NBP 88.8 88.4 85.8 83.5 71.8 62.0 52.2 37.7 the previous case, the degree of degradation was higher in NBP than DMF over a longer period Fmoc-Arg(NO )-OH 2 100 100 100 100 100 (Figure(DMF 2). or NBP) 1 Fmoc-Arg(Pbf)-OH Table 1. Stability of Fmoc-Arg(X)-OH100 100 in DMF 100 (N,N- 100dimethylformamide) 100 and NBP (N- (DMF or NBP) 1 butylpyrrolidone) at room temperature. 1 The study was stopped after 10 days, no changes were observed during this period. 0 h 1 h 24 h 48 h 10 d 15 d 20 d 30 d Fmoc-Arg(Boc)2-OH, DMF 88.8 88.6 86.9 85.0 77.6 65.1 58.5 51.2 Stability was examined again at 45 ◦C in DMF and NBP, in the presence of OxymaPure, Fmoc-Arg(Boc)2-OH, NBP 88.8 88.4 85.8 83.5 71.8 62.0 52.2 37.7 which are conditions commonly used in SPPS. Again, Pbf and NO2 analogues showed total stability Fmoc-Arg(NO2)-OH 100 100 100 100 100 (Figures S7–S10),(DMF while or NBP) the1 bis Boc derivative degraded slightly faster than in the previous experiment, but could stillFmoc-Arg(Pbf)-OH be considered compatible with the standard reaction times for coupling (up to 4 h). As 100 100 100 100 100 in the previous(DMF case, or NBP) the degree1 of degradation was higher in NBP than DMF over a longer period (Figure2). 1The study was stopped after 10 days, no changes were observed during this period.

FigureFigure 2. 2.Stability Stability of of the the mixture mixture ofof Fmoc-Arg(Boc)Fmoc-Arg(Boc)22-OH/OxymaPure-OH/OxymaPure 1:1 1:1 in in DMF DMF (A) (A )and and NBP NBP (B) (B )at at 4545◦C. °C. Elution: Elution: 30-95% 30-95%of of BB intointo AA inin 1515 min.min.

2.2.2.2.δ -Lactamδ-Lactam Formation Formation TheThe formation formation of ofδ δ-lactam-lactam was was studiedstudied followingfollowing Scheme 11.. TheThe carboxylic groups groups of of the the three three protectedprotected Arg Arg derivatives derivatives were were activated activated usingusing N,N’-diisopropylcarbodiimide (DIC) (DIC) and and OxymaPure OxymaPure asas additive additive in in an an equimolar equimolar mixture mixture (1:1:1) (1:1:1) inin DMFDMF oror NBP at 45 °C.◦C. The The mixture mixture (1.5 (1.5 equiv.) equiv.) was was then then addedadded onto onto the peptidethe peptide H-Gly-Phe-Leu-NH-Rink-amide-polystyrene-resin. H-Gly-Phe-Leu-NH-Rink-amide-polystyrene-resin. Aliquots Aliquots of the supernatant of the weresupernatant taken at diwereﬀerent taken times at different and analyzed times and by HPLC.analyzed After by HPLC. 120 min After of coupling, 120 min of the coupling, resin was the ﬁltered, resin washedwas filtered, and Fmoc washed removed and Fmoc before removed cleavage before and cleavage total deprotection and total deprotection (except in case (except of the in NOcase2 ofgroup) the usingNO2 highgroup) TFA-triisopropylsilane using high TFA-triisopropylsilane (TIS)-H2O (95:2.5:2.5). (TIS)-H2O The(95:2.5:2.5). crude peptides The crude were peptides then analyzed were then by HPLCanalyzed [24]. by HPLC [24].

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Scheme 1. Activation of protected Arg derivatives andand incorporation on a tripeptidyl resin.

The analysis analysis of of the the supernatants supernatants of each of each derivative derivative revealed revealed that DMF that DMFand NBP and showed NBP showed a similar a similarbehavior behavior in the three in the protecting three protecting groups. groups. More im Moreportantly, importantly, the same the analysis same analysis showed showed that Fmoc- that Fmoc-Arg(NOArg(NO2)-OH 2had)-OH the had least the tendency least tendency to form to formδ-lactam.δ-lactam. There There was a was good a good fertile fertile consumption consumption (the (thedecrease decrease in protected in protected Arg Arg was was not nottranslated translated into into the the formation formation of ofδ-lactam),δ-lactam),1 because (In the context at 30 min of thisthe manuscript,75% decrease “fertile in Fmoc-Arg(NO consumption”2)-OH refers2 was to thetranslated decrease into in theonly presence 3% of δ-lactam, of the protected having approximately Arg derivative when15% of it active remains ester as an (Figure active ester3A). This after finding activation imp orlies acylates that the the rest peptide of the resin) monomer because was at 30 incorporated min the 75% decreaseinto the peptidyl in Fmoc-Arg(NO resin, which2)-OH was (It corroborated is also important by high to consider coupling that (>99% part ofyield). the protected For Fmoc-Arg(Pbf)- Arg shown afterOH, the 30 minkinetics could was derive slower, from except the hydrolysis for δ-lactam of the formation corresponding (Figure active3B). Thus, ester at and 30 thereforemin, the formation would be partof δ-lactam of the “fertile was greater consumption”.) (12%, four was times translated more) than into onlyfor the 3% NO of δ2 -lactam,derivative. having Furthermore, approximately although 15% ofthe active fertile ester consumption (Figure3A). of Thisthe initial finding Fmoc-Arg(Pbf)-OH implies that the rest (40% of thedecrease monomer of the was initial incorporated protected intoArg2 theand peptidyl8% formation resin, of which the active was corroboratedester) was less by than high that coupling achieved (> 99%by the yield). NO2 analogue, For Fmoc-Arg(Pbf)-OH, the result was thethat kinetics after 120 was min slower, it yielded except same for δcoupling-lactam formationefficiency (Figure(>99%).3 Finally,B). Thus, Fmoc-Arg(Boc) at 30 min, the2 formation-OH showed of δthe-lactam fastest was kinetics greater of (12%, δ-lactam four timesformation more) (60%) than for(Figure the NO 3C),2 derivative. which translated Furthermore, into low although coupling the 2 fertileefficiency consumption (28%) as a of result the initial of the Fmoc-Arg(Pbf)-OH side-reaction. Overall, (40% the decrease lower of tendency the initial of protected the NO2 analogue Arg and 8%vs. formationthe (Boc)2 ofand the Pbf active analogues ester) was to lessrender than δ-lactam that achieved is reflected by the by NO the2 analogue, observation the resultthat the was important that after 120increment min it yieldedof the side-product same coupling occurred efficiency after ( 60>99%). min in Finally, NO2, when Fmoc-Arg(Boc) presumably2-OH the showedcoupling the had fastest been kineticscompleted of δand-lactam therefore formation the active (60%) ester (Figure could3C), no which longer translated be fertile. into Even low at coupling120 min, a e ffiratiociency of almost (28%) as1:1 aof result δ-lactam of the and side-reaction. active ester Overall,was present. the lower In the tendency case of Pbf of the analogue NO2 analogue at 120 min, vs. thealmost (Boc) all2 and the Pbfactive analogues ester had to been render convertedδ-lactam to is the reflected side-product. by the observation Finally, in thatthe case the importantof the (Boc) increment2 analogue, of the side-productactive ester was occurred not present after 60at any min time, in NO only2, when the δ presumably-lactam was the found. coupling had been completed and therefore the active ester could no longer be fertile. Even at 120 min, a ratio of almost 1:1 of δ-lactam and active ester was present. In the case of Pbf analogue at 120 min, almost all the active ester had been converted to the side-product. Finally, in the case of the (Boc)2 analogue, the active ester was not present at any time, only the δ-lactam was found.

1 In the context of this manuscript, “fertile consumption” refers to the decrease in the presence of the protected Arg derivative when it remains as an active ester after activation or acylates the peptide resin. 2 It is also important to consider that part of the protected Arg shown after 30 min could derive from the hydrolysis of the corresponding active ester and therefore would be part of the “fertile consumption”.

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FigureFigure 3. 3. High-performanceHigh-performance liquid liquid chromatography chromatography (HPLC) (HPLC) chromatograms chromatograms of the supernatants of the supernatants after

afterthe theprotected protected Arg Argderivatives derivatives [A, NO [A2;, B NO, Pbf;2; BC,, Pbf;(Boc)2C] ,were (Boc) activated2] were with activated DIC ( withN,N’- DIC (N,N’-diisopropylcarbodiimide)diisopropylcarbodiimide) and OxymaPure and OxymaPure and then andadded then to the added peptidyl to the resin. peptidyl Elution: resin. 30–95% Elution: of 30–95%B into ofA in B into15 min. A in 15 min.

2.3.2.3. On-Resin On-Resin Removal Removal of of NONO22 from the the Arg Arg Side-Chain Side-Chain

Once the suitability of the NO2 derivative of the Arg had been demonstrated in terms of stability Once the suitability of the NO2 derivative of the Arg had been demonstrated in terms of stability andand the the low low tendency tendency of δof-lactam δ-lactam formation, formation, our our attention attention turned turned to theto developmentthe development of a of new a new removal methodremoval with method broad with applicability broad applicability and easy and handling. easy handling. Inspired Inspired by our by previous our previous work work on the on removal the removal of p-nitrobenzyloxycarbonyl (pNZ) as protecting group of amines in SPPS [33], the capacity of p-nitrobenzyloxycarbonyl (pNZ) as protecting group of amines in SPPS [33], the capacity of SnCl2 of SnCl2 in a slightly acid medium to remove the NO2 group was investigated. A similar method has in a slightly acid medium to remove the NO group was investigated. A similar method has been been extensively used in the early times of comb2 inatorial chemistry to reduce polymer-supported extensively used in the early times of combinatorial chemistry to reduce polymer-supported nitro nitro aromatic compounds [34,35]. Taking the tripeptide H-Leu-Arg(NO2)-Phe-NH-Rink-amide-resin aromaticas a model, compounds an initial [ 34fine-tune,35]. Taking of the the removal tripeptide strategy H-Leu-Arg(NO was carried 2out.)-Phe-NH-Rink-amide-resin The conditions were then as a model,further an adjusted initial fine-tune with the peptid of theyl removal resins shown strategy in Figure was carried 4. The out.list included The conditions the lineal were precursor then furtherof adjustedCilengitide with and the peptidylbradykinin, resins two shownpeptides in of Figure biological4. The interest, list included and also the two lineal peptides precursor containing of Cilengitide the andArg-Trp bradykinin, sequence two repeated peptides twice of and biological three times. interest, These and molecules also two thus peptides allowed containingus to study the the effect Arg-Trp sequenceof an increasing repeated number twice andof NO three2 groups times. to be These removed, molecules as well thus as the allowed effect of us the to removal study theconditions effect of an increasing number of NO2 groups to be removed, as well as the effect of the removal conditions on a Int. J. Mol. Sci. 2020, 21, 4464 7 of 12

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 12 fragile residue like Trp. As references, the same peptides were synthesized using Fmoc-Arg(Pbf)-OH. Allon a peptides fragile residue synthesized like Trp. with As references, NO2- or Pbf-protecting the same peptides groups were were synthesized cleaved using from Fmoc-Arg(Pbf)- the resin using TFA-TIS-HOH. All peptides2O (95:2.5:2.5) synthesized for 1 with h at r.t.NO2- or Pbf-protecting groups were cleaved from the resin using TFA-TIS-H2O (95:2.5:2.5) for 1 h at r.t.

Figure 4. Peptidyl resins used in this study.

As a starting starting point, point, using using the the H-Leu-Arg(NO H-Leu-Arg(NO2)-Phe-NH-Rink-amide-resi2)-Phe-NH-Rink-amide-resinn as substrate as substrate and andthe theconditions conditions developed developed by our by group our group to remove to remove pNZ pNZ(solutio (solutionsns of 6–8 of M 6–8 SnCl M2, SnCl 0.4 M2, 0.4phenol, M phenol, either either0.064 M 0.064 HCl-dioxane M HCl-dioxane or 0.0016 or M 0.0016 AcOH M in AcOH DMF in at DMF55 °C), at the 55 ◦followingC), the following parameters parameters were studied: were studied:concentration concentration of SnCl2 (taking of SnCl into2 consideration(taking into considerationthat 8 M SnCl2 is that a supersaturate 8 M SnCl2 solutionis a supersaturate of difficult solutionhandling), of the di ffineedcult of handling), phenol, the the best need acid of rectif phenol,ier and the its best concentration, acid rectifier the and temperature, its concentration, and the thesolvent temperature, (SI, Table and1). The the results solvent can (SI, be Table summarized1). The results as follows: can be (i) summarized after assaying as DMF, follows: DCM/DMF, (i) after assayingand the DMF, green DCM solvents/DMF, and MeOH, the green EtOH, solvents NBP, MeOH, cyclopentylmethyl EtOH, NBP,cyclopentylmethyl ether (CPME), ether (CPME),and 2- andmethyltetrahydrofurane 2-methyltetrahydrofurane (2Me-THF), (2Me-THF), the best the results best results were found were found with the with latter. the latter. While While DMF DMFgave gaveacceptable acceptable results, results, NBP NBPwas, was,on this on occasion, this occasion, less suitable. less suitable. The use The of use EtOH of EtOH gavegave slower slower kinetics kinetics and andMeOH MeOH and andCPME CPME yielded yielded the poorest the poorest results, results, probably probably due dueto its to poor its poor capacity capacity to swell to swell the theresin; resin; (ii) (ii)2 M 2 SnCl M SnCl2 rendered2 rendered similar similar (or (or even even better) better) performance performance than than 6-8 6-8 M M solutions solutions and and therefore therefore is the concentration preferred;preferred; (iii) (iii) the the absence absence of of phenol phenol gave gave slightly slightly poorer poorer results; results; and and (iv) (iv) the the use use of 0.2of 0.2 M aqM HCl,aq HCl, which which is easier is toeasier use, provedto use, toproved be good to substitute be good forsubstitute HCl-dioxane. for HCl-dioxane. This final concentration This final ofconcentration acid is compatible of acid withis compatible the presence with of the acid-labile presence protecting of acid-labile groups protecting and it does groups not and cause it cleavagedoes not fromcause the cleavage resin since from it the represents resin since only it 0.64% represents of the on solution.ly 0.64% Additionally, of the solution. in some Additionally, of these experiments, in some of itthese was experiments, observed that it the was kinetics observed during that the the first kinetics hour wereduring very the slow, first andhour that were the very reaction slow, was and faster that afterthe reaction addition was of faster fresh after solution. addition Therefore, of fresh a solu previoustion. Therefore, washing witha previous a solution washing of 0.2 with M aqa solution HCl in 2-MeTHFof 0.2 M aq was HCl carried in 2-MeTHF out before was the carried removal out treatment. before the This removal approach treatment. greatly This enhanced approach the removal. greatly Thisenhanced acidic the washing removal. can This neutralize acidic traceswashing of the can piperidine neutralize used traces to of remove the piperidine the Fmoc used group to at remove the end the of theFmoc peptide group chain at the elongation. end of the As apeptide result of chain this preliminary elongation. set As of experiments,a result of this washings preliminary with 0.2 set M aqof HClexperiments, in 2-MeTHF washings (3 1 min) with followed 0.2 M aq by HCl 2 M SnClin 2-MeTHF-0.04 M phenol-0.2(3 x 1 min) M followed aq HCl in by 2-MeTHF 2 M SnCl (2–32-0.041 M h) × 2 × atphenol-0.2 55 ◦C were M consideredaq HCl in 2-MeTHF optimal conditions (2-3 x 1 h) for at 55 the °C LRF were model considered sequence optimal (Figure conditions5A). These for conditions the LRF weremodel then sequence applied (Figure successfully 5A). These to the conditions RGD peptides were then (Figure applied5B). Interestingly, successfully theto the removal RGD peptides kinetics for(Figure the RGD 5B). Interestingly, pentapeptide the were removal much fasterkinetics than for forthe the RGD LRF pentapeptide tripeptide. Afterwere amuch single faster treatment than for of 30the min, LRF the tripeptide. RGD peptide After showed a single more treatment than 97% of deprotection30 min, the whilstRGD peptide only 30% showed was observed more than in case 97% of LRF.deprotection This finding whilst prompted only 30% us was to do observed a new assay in case for of this LRF. peptide This reducingfinding prompted the amount us ofto SnCldo a2 newto a concentrationassay for this ofpeptide 1M, in reducing which the the total amount deprotection of SnCl was2 to achieveda concentration after 3 of30 1M, min in treatment which the (Figure total × S11).deprotection This result was suggested achieved thatafter the3 x removal 30 min oftreatment the NO 2(Figuregroup couldS11). This be sequence-dependent, result suggested that thus the potentiallyremoval of facilitatingthe NO2 group theuse could of evenbe sequence-dependent, milder conditions than thus those potentially generally facilitatin proposedg the in use some of cases.even milder conditions than those generally proposed in some cases.

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FigureFigure 5. 5.5.HPLC HPLCHPLC of of of the the the removalremoval removal of of the the NO NONO22 group2 group from from from H-Leu-Arg(NO H-Leu-Arg(NO H-Leu-Arg(NO2)-Phe-NH-Rinkamide-resin2)-Phe-NH-Rinkamide-resin2)-Phe-NH-Rinkamide-resin (A ()A ) (Aand) and H-Asp(OtBu)-Phe-Gly-Arg(NOH-Asp(OtBu)-Phe-Gly-Arg(NO H-Asp(OtBu)-Phe-Gly-Arg(NO2)-Gly-NH-Rink-amide-resin2)-Gly-NH-Rink-amide-resin2)-Gly-NH-Rink-amide-resin (B ()B using) using (B) using2 2M M SnCl SnCl 2 M2-0.042 SnCl-0.04 M2 M -0.04phenol- phenol- M phenol-0.20.2 MM aqaq HClHCl M aq inin HCl 2-MeTHF2-MeTHF in 2-MeTHF at at 55 55 °C. °C. at a: a: 55 protected protected◦C. a: protected peptide; peptide; b: peptide;b: unprotected unprotectedb: unprotected peptide. peptide. Elution: Elution: peptide. 10-25% 10-25% Elution: of ofB B 10-25%intointo AA of forfor B into((AA)) andand A for 5-95%5-95% (A) andof of B B 5-95%into into A A of for for B ( intoB(B) )in in A 15 15 for min. min. (B) in 15 min.

Next,Next, these thesethese conditions conditionsconditions were werewere applied appliedapplied to toto remove remove remove the the the two two two NO NO NO2 2groups 2groups groups ofof of bradykinin.bradykinin. bradykinin. On On this this occasion,occasion, the thethe kinetics kineticskinetics was waswas slower slowerslower than than inthan the in precedingin thethe precedingpreceding cases. Incases. cases. the searchIn In the the for search search alternatives for for alternatives alternatives to accelerate to to theaccelerate reaction, thethe two reaction,reaction, new assays twotwo new new were assays assays then were were run, then onethen inru run, ann, one one ultrasonic in in an an ultrasonic ultrasonic bath (Figure bath bath (Figure S12) (Figure and S12) S12) the and and other the the underother microwave under microwavemicrowave (MW) (MW) (Figure(MW) (Figure (Figure6). Both 6). 6). Both renderedBoth rendered rendered the the expected the expected expected results, results, results, namely namely namely a a faster fastera faster reaction, reaction, reaction, withwith ultrasound ultrasound leading leading to to total total deprotection deprotection in in 3 3 x1 1 hh treatmentstreatments (as(as inin thethe casecase of the LRF peptide) with ultrasound leading to total deprotection in ×3 x 1 h treatments (as in the case of the LRF peptide) andand MW MW in in 3 3 x30 30 min min treatments. treatments. AlthoughAlthough MWMW waswas faster,faster, thethe ultrasonicultrasonic bath may be a more and MW in ×3 x 30 min treatments. Although MW was faster, the ultrasonic bath may be a more accessibleaccessible device. device.device.

Figure 6. HPLC of the removal of the NO groups from protected bradykinin-NH-Rink-amide-resin Figure 6. HPLC of the removal of the NO2 2 groups from protected bradykinin-NH-Rink-amide-resin Figure 6. HPLC of the removal of the NO2 groups from protected bradykinin-NH-Rink-amide-resina: usingusing 2 2 M M SnCl SnCl2-0.042-0.04 MM phenol-0.2phenol-0.2 MM aq HCl in in 2-MeTHF 2-MeTHF at at 55 55 °C◦C (MW). (MW). a: protectedprotected peptide; peptide; b: using 2 M SnCl2-0.04 M phenol-0.2 M aq HCl in 2-MeTHF at 55 °C (MW). a: protected peptide; b: b:unprotectedunprotected peptide. peptide. Elution: Elution: 5–95% 5–95% of of B Binto into A Ain in 15 15 min. min. unprotected peptide. Elution: 5–95% of B into A in 15 min. Finally, to continue studying the removal of multiple NO groups, (RW)nP-NH (n = 2 or 3) Finally, to continue studying the removal of multiple NO2 groups,2 (RW)nP-NH2 (n 2= 2 or 3) were Finally, to continue studying the removal of multiple NO2 groups, (RW)nP-NH2 (n = 2 or 3) were weresynthesized. synthesized. The Theconditions conditions applied applied were were the general the general ones ones used used previously previously under under sonication. sonication. For synthesized. The conditions applied were the general ones used previously under sonication. For Forboth both peptides, peptides, the the results results obtained obtained after after 3 3x 1 1h h treatments treatments were were highly highly satisfactory satisfactory and and more than both peptides, the results obtained after 3 x× 1 h treatments were highly satisfactory and more than acceptableacceptable (Figure (Figure7). 7). acceptable (Figure 7).

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Figure 7. HPLC of the removal of the NO2 groups from H-[Trp(Boc)-Arg(NO2)]2-Pro-NH-Rink-amide Figure 7. HPLC of the removal of the NO2 groups from H-[Trp(Boc)-Arg(NO2)]2-Pro-NH-Rink-amide resin (A) and H-[Trp(Boc)-Arg(NO2)]3-Pro-NH-Rink-amide resin (B) using 2 M SnCl2-0.04 M phenol-0.2 resin (A) and H-[Trp(Boc)-Arg(NO2)]3-Pro-NH-Rink-amide resin (B) using 2 M SnCl2-0.04 M phenol- M aq HCl in 2-MeTHF at 55 C. a: protected peptide; b: unprotected peptide. elution: 5-95% of B into 0.2 M aq HCl in 2-MeTHF at◦ 55 °C. a: protected peptide; b: unprotected peptide. elution: 5-95% of B A in 15 min. into A in 15 min. 3. Conclusions Conclusion Arg is a key amino acid for the construction of peptides with relevant biological activity. Its guanidinoArg is a side-chainkey amino requiresacid for the protection construction to avoid of peptides side-reactions with relevant and to facilitatebiological its activity. solubility. Its Pbf,guanidino which side-chain is the most requires widely protection used protecting to avoid group, side-reactions shows some and to drawbacks, facilitate its mainly solubility.δ-lactam Pbf, formation,which is the stability most widely to the used TFA-based protecting global group, deprotection shows some conditions, drawbacks, and mainly a high δ-lactam price, formation, the latter duestability mainly to the to theTFA-based preparation global of deprotection the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl conditions, and a high price, the latter due mainly chloride to intermediatethe preparation and of its the posterior 2,2,4,6,7-pentamethyldihydro incorporation into thebenzofuran-5-sulfonyl guanidino group. chloride intermediate and its posterior incorporation into the guanidino group. The NO2 group, which was first described by Bergmann, has been scarcely used in SPPS mainly becauseThe it NO has2 group, to be removed which was by first catalytic described hydrogenation by Bergmann, in a has post-cleavage been scarcely step used and in thisSPPS does mainly not alwaysbecause provide it has to satisfactory be removed results. by catalytic Furthermore, hydrogenation there has in been a post-cleavage some discrepancy step and in this the literaturedoes not always provide satisfactory results. Furthermore, there has been some discrepancy in the literature regarding the potential of the NO2 group to suppress δ-lactam. Herein, we demonstrated that the regarding the potential of the NO2 group to suppress δ-lactam. Herein, we demonstrated that the electron-withdrawing effect of the NO2 group minimizes its nucleophilicity, which is translated into a electron-withdrawing effect of the NO2 group minimizes its nucleophilicity, which is translated into reduction of the side-reaction when compared with Pbf protection. In contrast, bis-Boc protection is a reduction of the side-reaction when compared with Pbf protection. In contrast, bis-Boc protection highly prone to δ-lactam formation. Furthermore, this group showed limited stability in DMF and in is highly prone to δ-lactam formation. Furthermore, this group showed limited stability in DMF and OxymaPure containing DMF, while the NO2 and the Pbf were stable. in OxymaPure containing DMF, while the NO2 and the Pbf were stable. Importantly, here we report on the development of a new and straightforward method for removing Importantly, here we report on the development of a new and straightforward method for the NO2 group from Arg side chain while the protected peptide is still on the resin. This removal removing the NO2 group from Arg side chain while the protected peptide is still on the resin. This strategy is based on the use of SnCl2 as reducing agent in mild acid conditions using aq HCl as acid removal strategy is based on the use of SnCl2 as reducing agent in mild acid conditions using aq HCl rectifier in a solution of 2-MeTHF, which is a green solvent, at 55 C. As commonly found in peptide as acid rectifier in a solution of 2-MeTHF, which is a green solvent,◦ at 55 °C. As commonly found in chemistry, we observed that the removal reaction could be sequence-dependent. However, in the peptide chemistry, we observed that the removal reaction could be sequence-dependent. However, cases in which the removal is not efficient, the concourse of sonochemistry significantly accelerates the in the cases in which the removal is not efficient, the concourse of sonochemistry significantly removal reactions. The use of microwave heating also gave positive results. However, accessibility to accelerates the removal reactions. The use of microwave heating also gave positive results. However, this type of heating is limited in bioscience laboratories and therefore ultrasound baths emerge as the accessibility to this type of heating is limited in bioscience laboratories and therefore ultrasound baths method of choice. In some demanding sequences involving the unstable Trp residue, the appearance emerge as the method of choice. In some demanding sequences involving the unstable Trp residue, of small satellite peaks could be attributable to side-reactions, which should be further studied for the appearance of small satellite peaks could be attributable to side-reactions, which should be further each case. Although currently the price of the Fmoc-Arg(NO2)-OH is, in a 25-g scale, slightly more studied for each case. Although currently the price of the Fmoc-Arg(NO2)-OH is, in a 25-g scale, expensive than Fmoc-Arg(Pbf)-OH, in large-scale production the former should be more economical slightly more expensive than Fmoc-Arg(Pbf)-OH, in large-scale production the former should be due to it using less expensive raw materials. more economical due to it using less expensive raw materials. Overall, in terms of effectiveness and handling, Fmoc-Arg(NO2)-OH emerges as an interesting Overall, in terms of effectiveness and handling, Fmoc-Arg(NO2)-OH emerges as an interesting alternative to the existing Arg derivatives. alternative to the existing Arg derivatives.

3. Materials and Methods

3.1. Materials All reagents and solvents were from commercial suppliers and were used without further purification. Fmoc amino acids and Fmoc-Rink-amide AM PS resin (loading 0.74 mmol/g) were

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4. Materials and Methods

4.1. Materials All reagents and solvents were from commercial suppliers and were used without further purification. Fmoc amino acids and Fmoc-Rink-amide AM PS resin (loading 0.74 mmol/g) were purchased from Iris Biotech GMBH (Marktredwitz, Germany). DIC and OxymaPure were a gift from Luxembourg Biotech. (Ness Ziona, Israel) and N,N-diidsopropylethylamine (DIEA), SnCl2, and piperidine were supplied by Sigma-Aldrich (St. Louis, MO, USA). Organic solvents (DMF, CH2Cl2 (DCM)) and HPLC quality acetonitrile (CH3CN) were purchased from Merck (Kenilworth, NJ, USA). Milli-Q water was used for RP-HPLC analyses. Microwave treatments were carried using a Discover SP (CEM, Matthews, NC, USA). Scientech Ultrasonic Cleaner (Labotec, Durban, South Africa) was used for the ultrasound treatment. Analytical HPLC was performed on an Agilent 1100 system using a Phenomenex AerisTMC18 (3.6 µm, 4.6 150 mm) column, with flow rate of 1.0 mL/min and UV × detection at 220 nm. Chemstation software was used for data processing. Buffer A: 0.1% TFA in H2O; TM buffer B: 0.1% TFA in CH3CN. LC-MS was performed on Ultimate™ 3000, Aeris 3.6 µm Wide pore column, Phenomenex C18 (150 4.6 mm) column. × 4.2. Peptide Synthesis The peptides used in this study were synthesized manually using a syringe fitted with a porous polyethylene disc and attached to a vacuum trap for easy filtration. Synthesis were carried out 0.1 or 0.2 mmol scale Rink-amide-PS resin (loading = 0.74 mmol/g) using Fmoc/tBu protocols. For Fmoc removal, 20% of piperidine in DMF was used. Couplings were performed by 1.5 eq. of an equimolar mixture of Fmoc-amino acid derivative, DIC and Oxyma in DMF or NBP, which was allowed to react for 1 h. Global deprotection and cleavage was performed by adding TFA-TIS-H2O (95:2.5:2.5) to the peptidyl-resin for 1 h at rt. Then, chilled ether was added to precipitate the peptides and after centrifugation and decantation, the peptides were taken up in water. The crudes obtained were analyzed by HPLC and finally were lyophilized. The peptide identities were confirmed by LC-MS.

4.3. Stability Study Procedure Solutions (0.2 M) of each protected Arg analogue in the absence or presence of OxymaPure (0.2 M) were prepared and kept in sealed vials. Aliquots (10 µL) at diﬀerent times of each one were taken and diluted with CH3CN (490 µL), and 1µL of this mixture was injected in the HPLC.

4.4. δ-Lactam Formation An equimolar solution of Fmoc-Arg(X)-OH/DIC/OxymaPure (1.5 equiv.) in DMF or NBP was added to the tripeptidyl resin (H-GFL-resin) at 45 ◦C and kept reacting for 2 h. A 10-µL aliquot of the supernatant was taken from the mixture at 0, 30, 60, and 120 min and diluted to 0.5 mL with CH3CN. From this solution, 1 µL was injected and analyzed by RP-HPLC (30–95% B into A in 15 min). After 120 min, Fmoc was removed using 20% piperidine in DMF, and peptides were cleaved from the resin as described before. Amino acid coupling was quantiﬁed by integration of the peaks obtained in their HPLC (10–25% B into A in 15 min).

4.5. Removal of the NO2 Group

The removal solution was prepared by taking SnCl2 (1.8 g), phenol (4 mg) in 2-MeTHF (2 mL), then aqHCl (98 µL) was added and the mixture was sonicated until total solubility of SnCl2. The final volume was then made up to 5 mL using 2-MeTHF, which gave a 0.2 N concentration of HCl. Peptide resin (50 mg) was washed with a solution of 2-MeTHF-HClaq (0.1%) (1.5 mL, 2 1 min). × After filtration, the SnCl2 solution was added (1.5 mL) and the syringe was sealed. The reaction was then left for 30 min at 55 ◦C with sporadic stirring. The solution was filtered off and the resin was Int. J. Mol. Sci. 2020, 21, 4464 11 of 12

washed with 2-MeTHF. An aliquot was then taken for mini cleavage using TFA-TIS-H2O (95:2.5:2.5) (60 µL) as before. Fresh removal solution was added to the peptide resin and the process was repeated.

Supplementary Materials: The supplementary information is available online at http://www.mdpi.com/1422- 0067/21/12/4464/s1. Author Contributions: The work was designed by J.L., F.A, and B.G.d.l.T.; the experimental part was carried out by M.A. and A.K. under the supervision of F.A., and B.G.d.l.T. All authors contributed in the results and discussion. The first draft of the manuscript was prepared by M.A. and A.K. and all authors contributed to the final version. All authors have read and agreed to the published version of the manuscript. Funding: The work was funded in part by the following: the National Research Foundation (NRF) (# 105892 and Blue Sky’s Research Programme # 120386) and the University of KwaZulu-Natal (South Africa); the Spanish Ministry of Science, Innovation, and Universities (RTI2018-093831-B-100), and the Generalitat de Catalunya (2017 SGR 1439) (Spain). We thank to Dr. Peter White and Millipore-Sigma-Aldrich for supporting part of this research. We thank Dr. Jonathan A. Collins (CEM) for facilitating the use of the microwave instrument. Conflicts of Interest: The authors declare no conflict of interest.

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