α/β-Chimera peptide synthesis with cyclic β-sugar amino acids: the efficient coupling protocol

The synthesis of α/β-chimeras comprises peptide bond formation from α- to β-, from β- to β-, and from β- to α-amino acid residues. The fine-tuned solid phase synthesis of –GXXG– chimera peptides containing the simplest achiral α-amino acid glycine and two cyclic SAAs of different ring size [X denoting cyclic β-Sugar Amino Acids (β-SAA)] is reported, variants containing Fmoc–RibAFU(ip)–OH a furanoid-, and Fmoc–GlcAPU(Me)–OH a pyranoid-type structural “Lego-element”. Systematic search for the best coupling strategy with both H–β-SAA–OHs is described, including the comparison of the different coupling reagents and conditions. Selecting the optimal reagent (from commonly used PyBOP, HATU and HOBt) was assisted by time-resolved 1H-NMR: formation and stability of the Fmoc protected active esters were compared. We found that PyBOP is the best choice for successfully coupling both H–β-SAA–OH prototypes. The present comparative results open a reasonable route for building efficiently various –β-SAA– containing homo- and heterooligomers.


Introduction
Chimeric synthetic polypeptides containing β-amino acids have achieved increasing attention in foldamer chemistry and drug design, due to their prolonged resistance against proteases and their ability of forming designed nanostructures of reduced internal dynamics (Horne and Gellman 2008;Pilsl and Reiser 2011;Guichard and Huc 2011;Kiss and Fülöp 2014;Cabrele et al. 2014;Mándity and Fülöp 2015;Pohl et al. 2013). Cyclic β-amino acid residues-as conformationally restricted building blocks-are of special interest, as they have the same number of backbone torsional angles (ϕ and ψ) as α-peptides. These oligo-and polypeptides composed of cyclic β-amino acids have backbone folding properties primarily determined by local coniguration. Coniguration driven, ine-tuned conformational properties enhance their receptor-binding ability and macromolecule formation potential (Beke et al. 2004).
acid (ACPC), and 2-aminocyclohexanecarboxylic acid (ACHC) derivatives are now widely applied to construct chimeras (Giuliano et al. 2013;Torres et al. 2009Torres et al. , 2010Gorrea et al. 2012;Herradón and Seebach 1989;Kessler et al. 1995;Kiss et al. 2017). However, all three of the above compounds are highly hydrophobic and their hydrophilic cyclic analogues are barely known and thus used. During the last 20 years, both ive-and six-membered cyclic Sugar Amino Acids (H-SAA-OHs) appeared sporadically as building blocks (Risseeuw et al. 2013;Pandey et al. 2011;Giri et al. 2012;Long et al. 1999;Simone et al. 2005;Sharma et al. 2008Sharma et al. , 2011Andreini et al. 2009, Chakraborty et al. 1998, von Roedern and Kessler 1994, Feher-Voelger et al. 2014) (Fig. 1). H-SAA-OHs were considered hard to synthesize and costly to manufacture on the gram scale required. Recently, we have accomplished several consecutive steps, starting from D-glucose. The syntheses are ine-tuned, optimized, and ready for large-scale and cost-eicient total synthesis; the Fmoc-derivatives are now available on the gram scale (Nagy et al. 2017;Goldschmidt Gőz et al. 2018). Homo-and heterooligomers containing H-β-SAA-OHs were synthesized by us and others using diferent coupling reagents (BOP, HATU, and HOBt) and amino protection (Fmoc or Boc groups) either in solution or on solid phase (Gruner et al. 2002;Csordás et al. 2016;Chandrasekhar et al. 2004;Suhara et al. 2002). For example, the pentofuranoid Fmoc-RibAFU(ip)-OH (1) was combined with β-homoglycine to form β-chimera peptides using HATU/ collidine reagents on solid phase (Gruner et al. 2002). The coupling of the same H-β-SAA-OH with α-glycine was similarly tested with HATU/DIEA (Csordás et al. 2016), but the coupling eicacy of the sugar moiety needs to be proved. Homooligomers of diferent lengths were prepared from the derivatives of the C-3 epimer H-XylAFU(ip)-OH with EDCI/HOBt in solution (Chandrasekhar et al. 2004). The hexopyranoid N-tert-butyloxycarbonyl-3,4,6-tri-Oacetyl-β-D-glucosamine-1-carboxylic acid was also used to form related types of homooligomers (Suhara et al. 2002). The syntheses were performed using BOP/DIEA reagents and Boc/Bn protection in solution. Although several coupling reactions and conditions were probed, no systematic comparison of the diferent coupling reagents, strategies, and techniques were performed yet. The comprehensive analysis presented here with respect to the coupling conditions and eicacy of two diferent H-β-SAA-OH prototypes representing diferent ring sizes: 5-membered ring, 3,4-trans Fmoc-RibAFU(ip)-OH (1) (Nagy et al. 2017) and 6-membered ring, 4,5-trans Fmoc-GlcAPU(Me)-OH (2) (Goldschmidt Gőz et al. 2018) residues were introduced into appropriate -Gly-β-SAA-β-SAA-Gly-heterooligomers. Our aims were to probe the key steps of peptide bond formation between α-and β-, β-and β-, and β-and α-amino acids using various active ester formation methods. Both the active ester types and the right coupling conditions (T, p, solvent type, reaction time, etc.) were optimized. Here, we present a systematic study on the formation and stability ine-tuned by 1 H-NMR of the active esters from the furanoid and pyranoid carbohydrate moieties.

Results and discussion
Our comprehensive study was carried out using four different, though commonly used peptide coupling reagents, namely: (i) the HOBt/EDCI used by Chandrasekhar et al. for H-β-SAA-OH in solution (Chandrasekhar et al. 2004); (ii) the HOBt/DIC system, the most prevalent choice of the ield (Valeur and Bradley 2009); (iii) the PyBOP/DIEA reagent pair developed for coupling "diicult sequences" without the considerable risk of racemization (Albericio and Carpino 1997); (iv) the HATU/DIEA also applied for "diicult sequences" with the risk of racemization (Valeur and Bradley 2009;Montalbetti and Falque 2005), and thus, limiting the overall coupling times to less than 3 h (Scheme 1). α/β-Chimera peptide synthesis with cyclic β-sugar amino acids: the eicient coupling protocol 1 3

Formation and stability of β-sugar amino acid active esters
During SPPS, the active ester is in situ made to enhance the eicacy of the amide bond formation. It is presumed that the active ester formation occurs fast and quantitatively (conversion > 99%) (Albericio and Carpino 1997). For α-L-amino acid residues, conditions for active ester formation were optimized to complete it within a few minutes (Coste et al. 1990). As cyclic β-sugar amino acids are more compact with a -COOH group sterically hindered, coupling conditions (Fig. 1) were probed and described here for selected H-β-SAA-OHs, using alternative coupling reagents. For a quantitative characterization, time-resolved 1 H-NMR spectra were recorded, selected resonance frequencies were assigned, and the time needed of the active ester formation (t f ) and hydrolysis (t h ) was established from the integral-time diagrams (see Supporting Information). The solution of the furanoid (1) or pyranoid (2) prototypes was mixed with the appropriate coupling reagent (PyBOP, HATU, or HOBt in an equimolar ratio) in [D 7 ] DMF at T = 25 °C. At t = 0 min either DIEA (2 eqv.), DIC (1 eqv.), or EDCI (1 eqv.) was added to initiate the active ester formation and 1 H-NMR spectra were recorded after 10, 20, 30, 60 min, and so on for hours (and day) ( When the active ester formation with HOBt/EDCI/DIEA is very slow (t f = 960 min), the reaction is complete; however, when it is fast (t f = 20 min), conversion is poor (20%, Fmoc-GlcAPU(Me)-OH, 2), and in addition, the product decomposes upon formation, the degree of the decomposition was monitored by the formation of HOBt side-product, the 1 H-NMR signals of which appeared in the spectra: H A : 7.78 ppm, H B : 7.60 ppm, H D : 7.93 ppm. The active ester formation is slow (t f = ~ 60-120 min) when HOBt/ DIC is used and the conversion also remains low (50%, Fmoc-RibAFU(ip)-OH, 1); moreover, the starting uronic acid (1) is fully regained. Additional experiments revealed that a stable active ester cannot be formed with HOBt directly as it contains equimolar H 2 O, which, in principle, is enough for the complete hydrolysis of the nascent ester. Furthermore, HOBt decomposes if crystal water is removed by drying with molecular sieves etc. Thus, stabilized forms of HOBt had to be used. Reaction of Fmoc-GlcAPU(Me)-OH (2) with HATU/DIEA is slow (t f = 240 min) presenting an additional problem as racemization can occur after 3 h. We found the active ester formation of Fmoc-RibAFU(ip)-OH (1) with HATU/DIEA or PyBOP/DIEA is fast (t f = 20 min) and the product remains stable for more than 24 h. Similarly, Fmoc-GlcAPU(Me)-OH (2) reacts with PyBOP/DIEA quickly (t f = 10 min) forming an active ester intact and stable for over 6 h. Thus, PyBOP/DIEA seems to be the only solution good enough to use and to match conditions required for standard SPPS coupling.
Comparing the four coupling reagent pairs and the time needed for the active ester formation, signiicant diferences were found (Table 1). The mechanisms of active ester formation can explain these diferences. We found that the ratedetermining steps of the ester formation are diferent using the HATU/DIEA or PyBOP/DIEA pairs and the HOBt/ EDCI/DIEA or HOBt/DIC pairs. When PyBOP/DIEA (or HATU/DIEA) (Scheme 2a) was used, the deprotonation of the carboxylic acid (2) by DIEA occurred quickly. The ratedetermining step is the next when the carboxylate anion as a nucleophile attacks on the electrophilic center, namely on the P atom of the phosphonium moiety of PyBOP (or at the C atom of the amidinium moiety of HATU).
However, using HOBt/DIC (or HOBt/EDCI), a weaker base (DIC or EDCI) deprotonates the carboxylic acid (1). This ion-pair formation is likely slow enough to become the rate-determining reaction step (Scheme 2b).
Thus, the active ester formation was found to be faster with PyBOP/DIEA and HATU/DIEA (Table 1) compared to HOBt/EDCI/DIEA and HOBt/DIC, in line with their mechanism explained above. Both for the furanoid Fmoc-RibAFU(ip)-OH (1) and for the pyranoid prototype, Fmoc-GlcAPU(Me)-OH (2), active ester formation with PyBOP is fast: t f = ~ 20 min and t f = ~ 10 min, respectively. On the contrary, the reaction with HATU is considerably slower (t f = ~ 240 min); therefore, in that case, pre-activation is necessary to avoid racemization during the coupling (3 h).
Due to their difering electronic structure, nucleophile attacks may occur diferently at P and C centers: the P atom can form a trigonal bipyramid structure, clearly more favorable for a nucleophile attack (Fig. 3), than that of C atom.
In conclusion, the use of PyBOP/DIEA pair provides the most promising conditions-the active esters are (i) formed quickly, (ii) products are stable for hours-to reliably form amide bonds between α-and β-, β-and β-, and β-and α-amino acid residues both in solution and on a solid support. α/β-Chimera peptide synthesis with cyclic β-sugar amino acids: the eicient coupling protocol 1 3 has to be taken into account as Gly has none and no racemization of the α-amino acid partner has to be considered as it is achiral. On the other hand, the bulky and cyclic H-β-SAA-OH residues which have several chiral C atoms, with -OHs diferently protected by large protecting groups, indicating considerable steric hindrance. Using SPPS and Fmoc chemistry, the synthesis of two -GXXG-tetrapeptides was completed, and thus, all three amide bond types probed, namely, the α-β, β-β, and β-α. Coupling conditions for both 1 and 2 (-RibAFU(ip)-and -GlcAPU(Me)-) were probed, and -GX-, -XX-, and -XG-amide bond formations were carefully monitored and competed (Tables 2, 3). Fmoc-β-SAA-OH 1 and 2 were synthesized according to our recently improved, multigram scale, environmentally friendly protocol (Scheme 3), starting from D-glucose and methyl α-D-glucopyranoside (Nagy et al. 2017;Goldschmidt Gőz et al. 2018). For difficult sequences, a low capacity resin (< 0.5 mmol/g) is recommended to be used. Therefore, RAM-Tentagel ® resin (0.24 mmol/g) for the Table 1 Time needed and conversion of the active ester formation and hydrolysis based on 1 H-NMR data recorded in DMF-d 7 (T = 25 °C) Recorded at 250 or 700 MHz, equimolar reagents were used (Fmoc-β-SAA-OH:reagent1:reagent2 or base) = 1:1:1 or 1:1:2 reaction conducted in an 5 mm NMR tube, and it was "shaken but not stirred"

3
Fmoc-GlcAPU(Me)-OH (2) and 2-chlorotrityl chloride (2-Cl-Trt-Cl) resin (an 1.6 mmol/g original capacity resin was tuned down to 0.25-0.36 mmol/g with the irst amino acid coupled to it) for the Fmoc-RibAFU(ip)-OH (1) was used. Coupling efficacy of the resin was completed by measuring its Fmoc capacity. The irst glycine moiety was linked to the resin in the usual manner via Fmoc-Gly-OH (Scheme 4), followed by the subsequent removal of Fmocprotecting group (see "Methods"). Coupling time need of our Fmoc-β-SAA-OHs (1 and 2) was optimized by 1 H-NMR as described above (Table 1). Coupling of both 1 and 2 was achieved to the free N-terminus of the irst Gly by using all four diferent coupling reagent pairs, namely: HATU/ DIEA, PyBOP/DIEA, HOBt/DIC and HOBt/EDCI/DIEA in DMF. Both the XX and the closing XG couplings were executed with the above four reagent types followed by the removal of the Fmoc protection (Tables 2, 3). Identiication of both -GXXG-tetrapeptides, 6 and 7, was done by ESI-MS ([M+H] + = 503 and [M+H] + = 870 (see in Supporting Information) once cleaved from the resins. Peptide 6 was cleaved with the mild AcOH:MeOH:DCM 1:1:8 cocktail to preserve the otherwise acid-labile isopropylidene O-protecting group of 1, while 7 with the milder 50% TFA (instead of the standard 95% TFA) to avoid the removal of O-benzyl protecting groups.
The eicacy of all four probed coupling reagent pairs was jointly evaluated (Tables 2, 3), and we found that coupling with HOBt/EDCI/DIEA is insuiciently moderate: 32% and 58% for the two SAAs, respectively. Despite of the longer reaction time needed (18 h coupling time) due to the slow active ester formation, we concluded that HOBt/EDCI/DIEA is unsuitable for producing chimera peptides with cyclic H-β-SAA-OHs on a solid support, in spite of the fact that it was successfully used for coupling of H-XylAFU(ip)-OH in solution (Chandrasekhar et al. 2004). Similarly to our previous results (Csordás et al. 2016) for HATU/DIEA reagent pair, we also achieved low eicacy: 34% and 46%, respectively. In addition, the use of HATU is problematic from yet another aspect: its coupling times might be too long to avoid racemization (< 3 h).

Conclusions
Solid-phase peptide synthesis conditions were optimized to effectively couple Fmoc-β-SAA-OHs (X = 1 and 2), prototypes of different ring size sugar amino acids, to A. Nagy et al.

3
produce chimera peptides. We found that the key step of success is the active ester formation. Therefore, we have focused both on reactivity and active ester stability by probing a furanoid (Fmoc-RibAFU(ip)-OH, 1) and a pyranoid (Fmoc-GlcAPU(Me)-OH, 2) β-amino acid derivative using the most common reagents, namely, HOBt/EDCI/DIEA, HATU/DIEA, PyBOP/DIEA, and HOBt/DIC. Using timeresolved 1 H-NMR measurements for deciphering the optimum conditions for -GX-, -XX-, and -XG-amide bond formations, and the diicult couplings of the -GXXG-α/βmodel peptides were successfully achieved with SPPS. We found the PyBOP/DIEA reagent pair to be the best among the probed coupling reagents, providing 80 to 100% coupling eicacy and an overall > 76% yield for both β-SAA containing tetrapeptides. Eicacy of the most hindered and thus most problematic -XX-bond formation with PyBOP/ DIEA can be as high as 91 or even 99%. The present comprehensive analysis has revealed that PyBOP may be the best choice to successful coupling H-β-SAA-OHs, providing now the possibility of making various β/β-homo-and α/β-heterooligomers for spectroscopic and pharmaceutical purposes.

Experimental section
Analytical data for all compounds (HPLC chromatograms, 1 H NMR, and MS spectra); 1 H NMR spectra, and igures of active ester formation of all coupling agents can be found in Supporting Information, in the online version.

Reagents and instrumentations
Reagents, materials, and solvents were obtained from Alfa Aesar, Sigma-Aldrich, Merck, Reanal, or VWR. For moisture-sensitive reactions, the solvents were distilled with the standard procedures or dried on molecular sieves (3 Ǻ). Products were analyzed by reverse-phase HPLC on a Phenomenex Jupiter C-18 column using the water/acetonitrile mixtures of 0.1% TFA in water (A) and 0.08% TFA, and 95% acetonitrile in water (B), and UV detection completed at 220 and 280 nm. Products were identiied with Bruker Esquire 3000+ tandem quadrupole mass spectrometer equipped with an electrospray ion source.

NMR measurements
1 H-NMR experiments were performed at 298-300 K on Bruker Avance DRX 250 MHz spectrometer equipped with 5-mm SB dual probe with z-gradient, operating at 250.13 MHz for 1 H and/or Bruker Avance III 700 MHz spectrometer operating at 700.17 MHz for 1 H equipped with 5-mm z-gradient probe head. Spectra were recorded in [D 7 ] DMF using the solvent residual peaks as the 1 H internal reference: 2.75, 2.93, and 8.03 ppm. The sample concentrations ranged from 10 to 20 mM. Spectra evaluation was completed within the TopSpin 3.5 software.

Peptide synthesis
For the SPPS using Fmoc-RibAFU(ip)-OH (1) 2-Cl-Trt-Cl resin, while for that with Fmoc-GlcAPU(Me)-OH (2) RAM-Tentagel ® resin was used. Resins were swollen in DCM. For the RAM-Tentagel ® resin, the irst step was α/β-Chimera peptide synthesis with cyclic β-sugar amino acids: the eicient coupling protocol 1 3 the removal of the Fmoc group with common method (2% piperidine and 2% DBU in DMF, 3 + 17 min). The successful cleavage was analyzed by the Kaiser test. Coupling of the Fmoc-Gly-OH to the 2-Cl-Trt-Cl resin was accomplished using Fmoc-Gly-OH (1.5 eqv. to the nominal capacity of the resin ~ 1.6 mmol/g) dissolved in DMF and DIEA (3.75 eqv.) was added to the solution: reaction lasted for 1 h. Coupling of the Fmoc-Gly-OH to RAM-Tentagel ® resin was made using Fmoc-Gly-OH (3 eqv. to the nominal capacity of the resin ~ 0.24 mmol/g) dissolved in DMF, HOBt (3 eqv.), and DIC (3 eqv.) was added to the solution: reaction lasted for 1 h. After coupling, the resins were washed with 3× DMF, 3× DCM, 3× MeOH and 1× diethyl ether and dried in vacuo. The capacity of the resin was determined by spectrometric measurement of the amount of Fmoc chromophore (Fmoc-piperidine adduct) released upon treatment of the resin with 20% piperidine in DMF (Chan and White 2000). The Fmoc group was removed by 2% piperidine and 2% DBU in DMF. The successful cleavage was analyzed by the Kaiser test.