Evaluation of 4-tert-Butyl-Benzhydrylamine Resin (BUBHAR) as an Alternative Solid Support for Peptide Synthesis

Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP, Brazil Department of Physiological Sciences, Santa Casa de Sao Paulo, School of Medical Sciences, 01221-020 Sao Paulo, SP, Brazil Department of Biochemistry and Chemistry Technology, Institute of Chemistry, Unesp, Araraquara, SP, Brazil Department of Biochemistry, Institute of Chemistry, Universidade de Sao Paulo, Sao Paulo, SP, Brazil


Introduction
Almost six decades after its inception, Merrifield's Nobel Prize solid-phase peptide synthesis (SPPS) method [1][2][3] continues to suffer from unresolved problems. Among these, there occur for instance great difficulties in some coupling reactions observed during the synthesis of strongly aggregating sequences. Usually site-dependent steric hindrance at the reactive N-terminal portion of a peptide segment is responsible for the impairment of coupling reactions. Alternative experimental approaches for minimizing this problem have been proposed in the literature [4,5], most of which have focused in modifying acylating agents [6,7] or reaction conditions [8,9].
Another option relies on the use of alternative resins with different solvent-dependent structural and bead solvation characteristics used mainly during the critical coupling reaction [10]. By following this line of investigation, we have already examined the feasibility of using resins with a very high degree of substitution (>2 mmol/g), obtained from a controlled experimental protocol [11,12]. More recently, alternative amine resins for the classical methylbenzhydrylamine resin (MBHAR) [13], used for the synthesis of α-carboxamide peptide in tert-butyl-(Boc-) SPPS chemistry, were introduced [14]. Among these, as an alternative to MBHAR (which contains a methyl group coupled to the phenylmethylaminefunctionalized copoly [styrene-divinilbenzene] structure), a larger more hydrophobic and electron-donating 4-tert-butyl group at this position (4-tert-butylbenzhydrylamine resin [BUBHAR]) was proposed. Still, in this report, other amine resins based on this same type of polymer matrix but containing chloro or dichloro groups as alternatives and known to be strong electron acceptor moieties were also proposed in order to examine the stability of peptide-resin linkage towards final acid cleavage step. In the case of BUBHAR, only preliminary tests were carried out with this solid support in which its feasibility for peptide synthesis was checked, based on the assembly of an easy-to-make sequence [14].
Thus, in order to better investigate the potential application of this alternative resin in the SPPS methodology, we decided to compare it with MBHAR to obtain α-carboxamide peptide through the Boc (tert-butyl) synthesis strategy. For this purpose, we decided initially to discard possible MBHAR suppliers production effects on its physical-chemical and structural properties. MBHAR batches (see Materials) were tested for comparison with those homemade BUBHARs in terms of swelling capacity of their beads accordingly to previous protocol [15][16][17]. In this case, the average volume of resin beads occupied by the solvent (in percentage) is calculated for estimating the solvation property of a resin in each solvent.
The next necessary step for this type of study involved a direct comparison between both resins with respect to overcoming a known difficult peptide synthesis step during the chain elongation of a peptide sequence. The selected hexapeptide Gln-Asn-Cys-Pro-(D-Arg)-Gly carboxy-terminal fragment of the antidiuretic hormone, desmopressin® (3-Mpa * -Tyr-Phe-Gln-Asn-Cys-Pro-(D-Arg)-Gly-amide), * 1-(3-mercaptoprionic acid) in which the Gln⟶Asn acylation step occurs only with a need for a recoupling reaction when MBHAR is used. Hence, the focus was now directed to this specific acylation in which solvation details of the Asn-Cys-Pro-(D-Arg)-Gly segment coupled to both resins were investigated in terms of their bead swelling capacities and also of degrees of peptide chain mobility spread throughout the polymer backbone of both resins when solvated in different solvent systems applicable for the SPPS method.

Synthesis of BUBHAR.
The general synthesis protocol of this type of polystyrene-divinylbenzene-based amino copolymer, initially described [13,19] and further modified by us [11] in order to better control the degree of substitution of amino groups in the resin, was used for preparing the three batches of BUBHAR. Basically, the amino group content of the resin was controlled at the Friedel-Crafts acylation step [11] followed by the Leuckart reduction reaction, which ensured the complete conversion of carbonyl groups generated in the previous step. The general procedure for the BUB-HAR synthesis is outlined using 4-tert-butylbenzoyl as the acylation agent in the Friedel-Crafts reaction. A general synthesis protocol for the synthesis of MBHAR and BUBHAR is shown in Scheme 1, below.   [15,17]. In short, 150-200 dry and swollen beads of each resin, allowed to solvate overnight, were spread over a microscope slide and the diameters of beads measured directly with an Olympus model SZ11 microscope coupled with Image-Pro Plus version 3.0.01.00 software. Since the size distributions in a sample of beads are log-normal rather than normal, the more accurate geometric mean values and geometric standard deviations were used in order to estimate the central value and the distribution of the particle diameters [20]. 2.2.7. EPR Studies. Electron paramagnetic resonance (EPR) measurements were carried out at 9.5 GHz on a Bruker ER 200SRC spectrometer at room temperature (22 ± 2°C) in flat quartz cells. Fmoc-Toac-labeled peptidyl-resins were preswollen overnight in the study solvent before the spectra were run, as previously introduced by us [21,22]. The magnetic field was modulated with amplitudes less than one-fifth of the line widths, and the microwave power was 5 mW in order to prevent saturation effects. The analyses were done in duplicate.  and amine group content values, BUBHAR's beads solvated better than those of MBHAR. Measured mean swelling values in this solvent system were 67% and 51% for BUBHAR and MBHAR, respectively, thus indicating better solvation properties for the tert-butyl instead of methyl groupattaching resin at position 4 of the phenylmethylamine moiety of the structure of both polymers.

Comparative Synthesis Study of a Model Peptide Segment in Both
Resins. The next step of the present study compared the efficiencies of the two resins in problems specifically related to the peptide synthesis methodology. In order to develop this second topic, as early explained, we decided to choose a difficult-to-couple amino acid reaction observed during the synthesis of the C-terminal hexapeptide Gln-Asn-Cys-Pro-(D-Arg)-Gly sequence of the antidiuretic desmopressin. In this peptide segment, we observed that irrespective of the source of MBHAR batch, its degree of substitution or acylation protocol used the Gln⟶Asn coupling reaction always needs to undergo the recoupling process. Thus, this critical coupling step in the desmopressin hormone was selected to more thoroughly evaluate the physicalchemical details of solvated BUBHAR and MBHAR solid supports attaching Asn-Cys-Pro-(D-Arg)-Gly sequence.
Batches of commercial MBHAR (0.7 mmol/g-Advanced ChemTech) and the homemade BUBHAR (0.75 mmol/g) were chosen as solid supports for synthesizing the Gln-Asn-Cys-Pro-(D-Arg)-Gly segment in 0.4 mmol synthesis scale. No synthesis problems were observed until the Asn residue incorporation, using standard coupling protocol which comprised the use of 2.5 molar excess of acylating components (amino acid and N,N`-diisopropylcarbodiimide (DIC)/4-hidroxybenzotriazole [HOBt]) in 50% DCM/DMF for 2 h at room temperature.
By using this approach, we detected a better solvation degree for the peptide-BUBHAR than for peptide-MBHAR support for all of the examined solvents. Moreover, the yield from both resins at this peptide sequence position, the swelling degree of peptide-resin beads in the routinely used 50% DCM : DMF solution for the acylation step was much greater for peptide-BUBHAR (83%) than for the peptide-MBHAR (46%), which is a relevant finding for the interpretation of the coupling reaction. Noteworthy and in accordance with these resin swelling data, there was no need for a Gln⟶Asn recoupling reaction in BUBHAR as systematically occurred with MBHAR. This result strongly supports the importance of the resin solvation property as previously demonstrated in critical acylation reactions within beads in other peptideresins [23][24][25].
(2) EPR Experiments with Spin-Labeled Peptide-Resins. In order to complement the above-described peptide-resin solvation, the degree of motion and steric hindrance neighboring of the peptide chains spread throughout the resin beads were now estimated by EPR experiments. Small portion of the N-terminal groups of peptide chains in both resins were labeled with the amino acid-type spin probe Toac (2,2,6,6tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) introduced by us for peptide and polymer labeling [26][27][28] as previously described for evaluating, for instance, the solvation properties of model peptide-polymers [21][22][23][24][25]. Figure 2 shows the EPR spectra of both peptide-resins in the solvent system (50% DCM: DMF) used for the coupling step of peptide synthesis in the present study, and Table 2 displays the EPR central peak line widths (W 0 ) values (in Gauss) that are known to be sensitive to the level of molecular mobility of labeled sites within resin network [16,22].
As a rule [16,25], there is an inverse correlation between the W 0 value and the rate of labeled molecular motion. Comparatively smaller W 0 values were obtained for peptide-BUBHAR than for peptide-MBHAR in all solvent systems, which were in close agreement with the swelling data of both peptide-resins.
Lastly, the analytical HPLC profiles of the crude Gln-Asn-Cys-Pro-(D-Arg)-Gly-peptide fragment assembled in both resins are shown in Figure 3. Rather similar profiles are observed but an interesting finding consisted of the presence of small contaminants near 13.26 min in the crude peptide cleaved only from MBHAR and after the Gln recoupling reaction in this resin (Figure 3(a)). To complement these In this case, the molecular weight found was 544.36 Da, which corresponds to the entire peptide sequence but lacking the Gln residue even after a double coupling of this residue.

Conclusions
The present report suggests that BUBHAR can be considered an efficient solid support for use in Boc-synthetic chemistry, and that it would be an alternative to the standard MBHAR. The better synthesis yields resulting from the use of BUB-HAR seems to be due to the improved solvation characteris-tics of its peptide-bearing polymeric matrix as demonstrated by experiments carried out not only with microscopic swelling measurements of peptide-resin beads but also to the greater peptide chain mobility that occurs exactly at the Gln-acylation step in this alternative solid support as verified with EPR experiments. In fact, these results seemed to be in close agreement with previous observations [14] in which much easier handling (swelling and washing of beads), regardless of the step of the SPPS synthetic cycle, was observed using BUBHAR than MBHAR. In order to deeper evaluate this comparative investigation between both resins, synthesis of the same peptide fragment is currently in progress, but using resin batches with a lower (~0.2 mmol/g) and higher (2.0-3.0 mmol/g) degree of amine group contents. Although designed to be used for the Boc-chemistry approach, the novel BUBHAR polymer might also be tested in the 9-fluorenylmethyloxycarbonyl-(Fmoc-) chemistry method [3,29] after previous coupling of appropriate spacer   5 International Journal of Polymer Science groups (linkers) necessary for peptide segment assembly. In this way, similarly to what was observed with other types of resins early proposed for use in SPPS [30,31], BUBHAR can be considered in the future, one more potentially usable for the SPPS methodology.

Data Availability
No data were used to support this study.

Conflicts of Interest
The authors of this manuscript declare no conflict of interest, either financial or otherwise.