Alternative and Simple Normal-Phase HPLC Enantioseparation of a Chiral Amino Acid-Type Spin Label Derivative

Neste trabalho desenvolveu-se um processo alternativo para separação cromatográfica dos enantiômeros (+)-(3R,4R) e (–)-(3S,4S) do β-aminoácido quiral trans-2,2,5,5-tetrametilpirrolidina3-amino-4-carboxílico (POAC), que estava protegido no grupo amínico para posterior ligação a um peptídeo, polímero ou outra macromolécula. A enantioseparação foi obtida por HPLC usando uma fase estacionária normal à base de celulose quiral e eluição isocrática. O sistema n-hexano:isopropanol, sempre com maior quantidade do primeiro solvente, foi usado como fase móvel, pois forneceu os melhores resultados na separação dos dois componentes, constatado pelos valores mais elevados de fator de separação e de índice de resolução cromatográfica. Estes parâmetros apresentaram valores de 3,7 e 18,4 e de 2,0 e 6,7 nas soluções com proporção 90:10 (v/v) e 80:20 (v/v) de n-hexano:isopropanol, respectivamente. Estes dados indicam que a estratégia de purificação cromatográfica em uma única etapa usando fase normal é viável, abrindo assim a perspectiva de uma produção rápida e em grande escala desta sonda paramagnética.


Introduction
In the early eighties, a chemical strategy for specifically and covalently coupling the paramagnetic amino acid derivative 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) 1 to the N-terminal end of a peptide using the solid phase method 2 was reported in the literature. 3,4About one decade later, an improvement in this experimental approach allowed the insertion of this spin probe into any internal position of the peptide backbone using the base-labile 9-fluorenylmethyloxycarbonyl (Fmoc)-N α temporary protecting group. 5Since the first synthesis of the Fmoc-TOAC derivative was described, a progressive increase in the use of this nitroxide-based achiral C αα -tetrasubstituted α-amino acid in electron paramagnetic resonance (EPR) analysis 6 has been observed in the literature as recently reviewed. 7][14] Although TOAC has a great potential, the low reactivity of its α-amine function is indeed a serious drawback when the subsequent amino acid of the peptide sequence has to be coupled. 4,15In an attempt to solve this problem, the 2,2,5,5-tetramethylpirrolidine-1-oxyl-3-amino-4carboxylic acid (POAC) spin probe, a β-amino acid described earlier 1 and derived with the Fmoc group, 16 was also proposed by us as an alternative paramagnetic probe for labeling peptide sequences. 17With the POAC probe, the coupling of the subsequent amino acid residue proceeded much faster than with TOAC, which comparatively required larger excess of reagents, repeated coupling reactions and use of more severe acylating conditions such as the increase in the reaction temperature. 5,15Unfortunately, POAC and its Fmoc-POAC derivative are chiral compounds due to the presence of two asymmetric carbon atoms (C 3 and C 4 ) in their pyrrolidine structure.On the other hand, previous X-ray diffraction studies 17 have shown that POAC is composed of only trans isomers, thus indicating that the main target is to develop a fast and practical chromatographic separation of its (+)-(3R,4R) and (-)-(3S,4S) enantiomers (Figure 1).
To date, this problem has been solved either by direct chromatographic separation of these enantiomers in an analytical chiral column (Chiralcel OD-RH) with application of a reversed-phase elution mode [0.1 mol L -1 aqueous KPF 6 /acetonitrile (80:20, v/v)] 18 or indirectly, by first synthesizing a POAC derivative with binaphthyl temporary group esterified to its carboxyl function [19][20][21] and then chromatographically separating the derivatized POAC molecule.After this step, both enantiomeric POAC derivatives are saponified to recover the parent Fmoc-POAC enantiomers.After this sequence of procedures, it is finally possible achieving complete enantioseparation of these two compounds to further use them in labeling of a peptide sequence. 22espite this successful result, the need for a fast and simple purification strategy that allows large-scale production of both Fmoc-POAC enantiomers for use in peptide synthesis still remains.The present study thus describes an alternative and practical strategy for enantioseparation of a recemic mixture of the Fmoc-POAC derivative.

Materials
All solvents and reagents and chemicals were of analytical grade and met the ACS standards.They were purchased from Aldrich or Sigma Co. (St.Louis, MO, USA).

LC/ESI-MS experiments
Electrospray ionization (ESI) operating in the positive ion detection mode was used for mass determination of enantiomers.The LC/ESI-MS experiments are performed on a system consisting of a separation module Waters Alliance model e2695 and a 2489 UV/Visible detector, both from Waters Systems (Milford, USA).This equipment is coupled to a mass detector Waters model 3100 and is controlled by a workstation ThinkCentre.The samples are automatically injected onto a Waters narrow bore Nova-Pak column C18 (2.1 x 150 mm, 60 Å pore size, 3.5 μm particle size).The elution is carried out with solvents A (0.1% TFA/H 2 O) and B (60% acetonitrile/0.1% TFA/H 2 O) at a flow rate of 0.4 mL min -1 using a linear gradient of 5-95% B in 30 min and UV detection at 220 nm.

EPR experiments
EPR spectra were acquired on a Bruker spectrometer (model ER 200D-SRC, room temperature).Fmoc-POAC samples (5 x 10 -5 mol L -1 ) were diluted in phosphate buffer (0.02 mol L -1 , pH 7.0; 22 ± 2 ºC) and placed in flat quartz cells for aqueous solutions (J.Scalon, Costa Mesa, CA, USA).Values of 0.5 G, 5 mW, 0.05 s and 9.5 GHz were used for modulation amplitude, microwave power, time constant and frequency parameters during EPR experiments.The spectra were collected with a field range of 100 G, centered approximately at 3450 G.The rotational correlation time (τ C ) values 23 were calculated as described elsewhere. 24 experiments CD spectra of the purified enantiomers (in methanol) were acquired in a Jasco spectropolarimeter (model 2095 Plus, Tokyo, Japan).Cylindrical quartz cells (0.1 mm path length) were used.

Results and discussion
The racemic trans-Fmoc-POAC probe was synthesized and characterized accordingly to above detailed protocols as earlier reported. 17HPLC separation of the (+)-(3R,4R) and (-)-(3S,4S) enantiomers was achieved using a chiral analytical column (Lux Cellulose-2, 250 mm length x 4.6 mm i.d.) containing tris-(3-chloro-4methylphenylcarbamate) cellulose-2 matrix as stationary phase.The mobile phase (n-hexane and isopropanol in different proportions) was used in a normal-phase mode isocratic-elution protocol.A constant flow rate (1 mL min -1 ) was used for elution of sample components.
Table 1 shows the values for retention time (t R ), separation factor (α), and resolution index (R s ) of both enantiomers 25,26 obtained with different proportions of the n-hexane:isopropanol mixture as mobile phase.In addition, Figure 2 shows the elution profiles obtained in different isocratic runs in which the proportion of n-hexane:isopropanol in the mobile phase varied from 10:90 (v/v) to 90:10 (v/v).It is noteworthy that an inversion occurs in the order of the eluting peaks.The (-)-(3S,4S) enantiomer eluting faster when the amount of n-hexane becomes higher than that of isopropanol.Optimized separation, as detected by the highest values for the chromatographic parameters α and R s , was observed with the 90:10 (v/v) proportion of n-hexane:isopropanol (Table 1 and Figure 2).
The 80:20 (v/v) solvent system also yields good results in terms of fractionation data of enantiomers, mainly when the time-consuming factor is also considered.Complete elution of both components is achieved after 7 min and 15 min for 80:20 (v/v) and 90:10 (v/v) mixed solutions, respectively (see Figure 2c vs. 2d).Despite this finding, the latter solution should be still taking into account in terms of practical effect as the mail goal of this study lies upon the search of an efficient separation of both enantiomers, mainly focused for application in large-scale protocol.In this case, the greatest separation observed between both Fmoc-POAC enantiomers (Figure 2d) with the 90:10 (v/v) n-hexane:isoproponal solvent can minimize the occurrence of a potential overlapping effect of peaks when larger amount of Fmoc-POAC has to be fractionated in the column.In this context, preliminary analytical chromatographic experiments increasing the flow rate (from 1.5 or 2.0 mL min -1 ) revealed problems of backpressure in the column (data not shown), possibly induced by the viscosity of the organic solvent systems.
For identification of each of the Fmoc-POAC enantiomers, they were first eluted as earlier reported, 18 using the chiral OD-RH Chiralcel column in HPLC reversed-phase mode.In this elution protocol, the (-)-(3S,4S) enantiomer, which eluted first in the Lux Cellulose-2 column (n-hexane:isopropanol, in 30:70 or 90:10, v/v), was in contrast, more retained in the column, thus eluting later than the (+)-(3R,4R) partner.Noteworthy, it was also possible to detect comparatively smaller peaks for the (-)-(3S,4S) enantiomer in the chromatogram.These findings should be due possibly to the observed lower solubility of this enantiomer in comparison with the (+)-(3R,4R) compound, inducing its precipitation/ aggregation during the different steps of the entire chromatographic fractionation procedure.
Mass spectra also confirmed the similar values for molecular weight of both enantiomers, and the EPR τ C values determined for these purified paramagnetic compounds (in methanol) were about 7 × 10 -11 s -1 .LC/ESI-MS and EPR spectra of both Fmoc-POAC enantiomers are displayed as Figure 1 and Figure 2, respectively, in the Supplementary Information.Finally, CD spectra of these compounds (in methanol) shows that the (+)-(3R,4R) and (-)-(3S,4S) enantiomers exhibit positive and negative Cotton effects in the region of 230 nm, respectively (Figure 3).
0][21] Otherwise, the use of a normal-phase elution protocol with the alternative Chiralcel OD-H column, whose selector is the same as that of the OD-RH column, failed in separating the enantiomers 18 with use of n-hexane:isopropanol mixture, the same mobile phase that was used in the present study with the Lux Cellulose-2 column.
A possible explanation for the successful use of normal-phase separation of the Fmoc-POAC enantiomers with a Lux Cellulose-2 column, in comparison with the Chiralcel OD-H column, might due to the presence of a chorine atom in the resin matrix [(tris-(3-chloro-4-methylphenylcarbamate) cellulose] of the former column.The high electronegativity of this atom would maximize selectivity due to a specific interaction between each enantiomer and the column stationary phase, thus favoring a better chromatographic resolution in the apolar environment provided by the n-hexane:isopropanol mixtures.Accordingly, the best resolution was observed with the 90:10 (v/v) and 80:20 (v/v) n-hexane:isopropanol mixtures, which yielded greater values of separation factor and resolution parameter (Table 1).
Indeed, besides the search for large-scale production strategy of Fmoc-POAC enantiomers, additional objectives also of this study comprise the improvement in the enantioseparation strategy of other β-amino acid-type compounds.Large amount of recent reports have shown that the incorporation of this type of organic compounds may generate relevant oligomers in the chemical and biological fields. 27,28In this sense, the investigation for alternative chromatographic purification strategies applicable specially for chiral products has resulted in publication of several studies 25,29,30 mainly targeting the isolation of chiral drugs with great potential for therapeutic purposes. 31,32

Conclusions
This study showed that enantioseparation of the racemic trans-Fmoc-POAC spin probe for application in the chemistry of peptides and other macromolecules is feasible.A simple and one-step normal-phase mode chromatography using a Lux Cellulose-2 column and n-hexane:isopropanol as mobile phase in appropriate proportion allowed easy isolation of the (+)-(3R,4R) and (-)-(3S,4S) enantiomers of this amino acid-type spin label.The results obtained with the 90:10 (v/v) or 80:20 (v/v) n-hexane:isopropanol mixtures exhibited high chromatographic resolutions with good separation factors and resolution indexes.In summary, an alternative and potentially useful HPLC enantioseparation, hopefully applicable for fast and large-scale chromatographic production of these two Fmoc-POAC enantiomers, was herein proposed for further application in the broad peptide and polymer fields.

Table 1 .
Chromatographic a Retention time; b separation factor; c resolution index.