Pseudo-enantiomeric coupling reagents for predictable incorporation into the peptide chain D and/or L amino acid residue of racemic substrates

Reaction of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) with bis-tetrafluoroborates of quinine and/or quinidine in the presence of sodium bicarbonate gave a pair of pseudo-enantiomeric coupling reagents: N , N ’ - bis-(4,6-dimethoxy-1,3,5-triazin-2-yl)quinine bis-tetrafluoroborate (2DMT/quinine/2BF 4) and N , N ’ -bis-(4,6-dimethoxy-1,3,5-triazin-2-yl)quinidine bis-tetrafluoroborate (2DMT/quinidine/2BF 4). The reagents activate the opposite enantiomers of racemic N -protected amino acids. By their reaction with two equivalents of racemic carboxylic components and diverse esters of amino acids, protected dipeptides were obtained with a predictable configuration and 95 – 99% enantiomeric homogeneity.


Introduction
Even a single modification of the configuration at numerous chiral centres in a peptide chain can dramatically impact the physicochemical properties, physiological activity, and degradation profiles of a peptide.The variations can be further multiplied by introducing non-proteinogenic building blocks into the peptide.Unfortunately, the search for structures offering the most desirable properties is extremely tedious, due to limited knowledge of structure-activity relationships.The quest for pharmaceutically valuable peptides is in most cases based on systematic synthesis of large numbers of analogues, involving diverse and often "exotic" building blocks.Access to both enantiomers of the chiral amino acid building blocks is therefore a crucial factor in the search.][9][10] These include kinetic resolution using chiral 4-dimethylaminopyridine (DMAP) analogues, [11][12][13][14] amidine based catalysts, 15 phosphines, [16][17][18] amines, 19 and others. 20,21heme 1. Attachment of rac-Pg-NH-Aaa*-COOH to the chiral amino-component H2N-Bbb*-COOR using chiral coupling reagent H-Y* proceeds with diastereoselectivity controlled by the three stereogenic centres already present in the substrates and an additional stereogenic centre in the tetrahedral intermediate.
Due to the specificity of the privileged scheme of peptide synthesis starting from the C-terminus, any enantioselective attachment of the subsequent building block rac-Pg-Aaa*-OH to the chiral amino-component H2N-Bbb*-OR using chiral coupling reagent H-Y* proceeds with diastereoselectivity controlled by the three stereogenic centres marked with an asterisk and by the additional stereogenic centre of the temporarily formed tetrahedral intermediate (Scheme 1).Under typical conditions of peptide bond formation, proceeding according to the AN*DN ‡ mechanism, 22 the yield, enantiomeric enrichment, and configurational preferences of each enantiomer of rac-Pg-NH-Aaa*-COOH for attachment to a distinct, usually complex, amino-component H2N-Bbb*-COOR can be predicted only after precisely optimized selection of the chiral coupling reagent H-Y* acting under strictly defined coupling conditions.Modifying the structure of any single coupling component requires subsequent optimization of all the parameters of the coupling process to obtain satisfactory results.Considering that in the peptide preparation, the amino-component H2N-Bbb*-COOR consists in almost all cases of complex, expensive, and highly diversified products of multistep synthesis, wasting it in an optimization procedure is unacceptable.
To overcome the problem of poor-predictability of peptide synthesis based on kinetic resolution, we designed binary enantioselective reagents for activating carboxylic components.According to the concept, the classic non-chiral triazine reagent with well-documented utility for peptide synthesis 23 was modified by introducing chiral module L*, which is responsible for enantioselective activation of the racemic carboxylic component and then departs after fulfilling its task. 24All further coupling stages can proceed under conditions optimized for the well-known classic non-chiral reagent.Thus, the preferred configuration and enantiomeric enrichment can be accurately predicted for any set of coupling components, via an experiment involving simple model substrates (Scheme 2).Scheme 2. Classic, non-chiral triazine coupling reagent with documented versatility in peptide synthesis was modified by replacing the N-methyl-1,4-morpholinium fragment with chiral leaving group L*.Fragment L*, responsible for enantioselective activation of the racemic carboxylic component, departs after fulfilling its task.
Reagents designed with chiral fragment L* prepared from strychnine or brucine gave fully predictable results of kinetic resolution.This allowed the synthesis of a representative group of enantiomerically enriched dipeptides from the racemic carboxylic component (used in two equivalents only), with 86-99 enantiomeric excess and 85-99% yields..25Moreover, in solid-state peptide synthesis using an excess of the racemic carboxylic component, final peptides were obtained with up to 100% enantiomeric homogeneity. 26owever, the versatility of the procedure based on the application of strychnine and/or brucine is limited by the fact that both alkaloids are available in only one configuration.
To overcome this limitation, several attempts were made to construct both enantiomeric forms of the chiral component L*.Esters of N-methylproline and N-allylproline are easily available in both enantiomeric forms.As expected, when applied as L*, these esters opened access to both enantiomeric Z-Ala-Gly-OMe dipeptides, but with only moderate yield (65-67%) and moderate ee of 70-74%. 27Attempts to improve enantioselective peptide synthesis were also made using reagents prepared from 2-chloro-4,6-dimethoxy-1,3,5triazine (CDMT, 3) and (5R) and (5S)-2-(trichloromethyl)-1-aza-3-oxabicyclo[3.3.0]octan-4-one.Kinetic resolution of the rac-Z-Ala-OH process gave both expected enantiomeric dipeptides, but with poor yields (13-19%). 28inchona alkaloids are extremely attractive for use as the chiral component L*, as they are readily available in two pairs of pseudo-enantiomeric forms.However, experiments involving treatment of quinine (1)  with CDMT (3) revealed that the primary reaction product, formed by the reaction of aromatic nitrogen of quinoline fragment with triazine reagent 3, was not active as a coupling reagent.Moreover, intensive degradation of quinine (1) was observed after the transfer of the triazine ring into the quinuclidine fragment followed by the opening the bicyclic system in a reaction with the nucleophilic chloride anion. 29Replacement of the chloride anion with a poorly nucleophilic tetrafluoroborate anion gave a stable quinine derivative with a triazine ring attached to the aromatic nitrogen of the quinoline fragment, but it was still not active as a coupling reagent.

Synthesis of coupling reagents
There are two tertiary quinine (1) nitrogen atoms prone to quaternization in reaction with CDMT (3).To avoid destruction of the quinuclidine bicyclic fragment by the nucleophilic chloride anion, 29 1 was treated with ammonium tetrafluoroborate obtaining quinine bis-tetrafluoroborate (2).The reaction of 2 with two equivalents of CDMT in the presence of sodium bicarbonate proceeded steadily.
Relatively fast consumption of 3 was observed in the preliminary phase of reaction with quinine bistetrafluoroborate (2), followed by a slower reaction in the more advanced stage of the process.

Peptide synthesis
The kinetic resolution of racemic carboxylic acid by enantioselective activation of the carboxylic function can be severely disturbed by the formation of meso-anhydrides, in a side-reaction of the rejected enantiomer with the activated enantiomer in the opposite configuration. 30To diminish the risk of this parasitic process, we made model experiments of 4-methoxybenzoic acid (8) activation with stoichiometric amounts of chiral coupling reagents 4 and 7 (Scheme 5).The coupling reagents were not completely consumed even after 24 h.Fortunately, the activation rate accelerated substantially after the addition of a tertiary amine to the mixture of 4methoxybenzoic acid and reagent 4 and/or 7. The results of optimizing the activation process depicted in Table 1 show that addition of 1, 5 or diisopropylethylamine (DIPEA) substantially accelerate the reaction rate, reducing the time necessary for consumption of the substrates to 2 h.In the case of coupling reagents 4 and 7. the highest coupling yields were obtained using as catalysts 30% of 1 or 5, respectively.In previous studies using reagents derived from strychnine and brucine, amino acids with aliphatic and aromatic amino acids were activated with opposite configurational preference. 24Therefore, in the present study on enantioselective activation rac-Z-Ala-OH (11) with an aliphatic side-chain and rac-Z-Phe-OH (12) with an aromatic side-chain were used.In both cases, two equivalents of 11 with an aliphatic side-chain and 12 with an aromatic side-chain were coupled with one equivalent of diverse amino components 13a-g in acetonitrile solution in the presence of 4 and 0.3 equivalents of quinine (1) used as catalyst.
Crude dipeptides 14a-g were isolated after evaporation of the reaction mixture to dryness, extraction of the residue with dichloromethane (DCM), successive washing of the extract according to the standard procedure with 1M HCl and saturated aqueous NaHCO3 solution, drying and evaporation again to dryness.Due to the ionic character of the substrates, by-products, and side-products, this isolation procedure gave more than 90% pure dipeptides while retaining the composition of stereoisomers.All signals expected for dipeptides 14a-g and 15bg were identified in the 1 H NMR and IR spectra of crude dipeptides prepared.In order to avoid modification of composition of the diastereoisomeric products by excessive purification procedure, the configuration and enantiomeric composition of the formed dipeptides 14a-m were determined by gas chromatography on chiral stationary phase after the hydrolysis of crude dipeptides to amino acids.
In all the couplings of aliphatic rac-Z-Ala-OH (two equivalents of 11) with methyl esters of amino acids 13a-g using reagent 4, the formation of D,L-dipeptides 14a-g was preferred (98-99.5% D,L), isolated with 56-60% yield (Table 2).Reversed preferences were found when two equivalents of aromatic rac-Z-Phe-OH (12) were coupled with methyl esters of L-amino acids 13b-g in the presence of reagent 4 (Table 3).In all cases, the preferred dipeptides 15b-g configuration was L,L (97% up to 99.5%), isolated with 56-62% yield.
Dipeptides with opposite configurations were obtained in enantioselective coupling using quinidine reagent 2DMT/quinidine/2BF4 (7) (Scheme 7).In all dipeptide syntheses from racemic aliphatic rac-Z-Ala-OH (11), seven different L-amino components 13a-g and quinidine reagent 7, the configuration of the preferred product 14a-g was L,L (98-99.5%)(Table 4).(11) with methyl esters of amino acids (13a-g) and two equivalents of rac-Z-Phe-OH (12) with methyl esters of amino acids (13b-g) in the presence of 2DMT/quinidine/2BF4 (7) (one equivalent).Again, reversed configurational preferences were found in the syntheses of dipeptides 15b-g from aromatic rac-Z-Phe-OH (11) and methyl esters of L-amino acids 13b-g (Table 5).In all cases, the configuration of the main products 15b-g was D,L (98-99.5%).In the cases of both reagents 4 and 7, excellent enantiomeric enrichment of the isolated dipeptides was accompanied by a relatively moderate yields in the range of 57-65%.
However, this limitation can be efficiently overcome by the application of excess of the acylating component, as is routinely practiced in solid-phase peptide synthesis.The stimulus of the inversion of configurational preferences in the acylation reactions involving Z-Phe-OH (11) activated by coupling reagent 4 prepared from quinine as well as coupling reagent 7 derived from quinidine still remain unrecognized.It can be presumed, however, that interactions involving the aromatic ring of the benzyl side-chain in phenylalanine, with quinine and quinidine fragments of coupling reagent, dominate over steric hindrance effects of the aliphatic alanine side-chain.

Conclusions
The application of pseudo-enantiomeric coupling reagents 4 and 7 derived from quinine (1) and quinidine (5)  respectively, opened a route to the incorporation of an amino acid building block with a predictable favoured configuration using a racemic substrate.The configuration of the reaction product as well the preferred reaction conditions can be accurately predicted after a single model experiment.Enantioselectivity in the range of 97-99.5% was obtained for both enantiomers, creating a brand-new tool for systematic studies of the relationship between the broadly defined properties of peptides and the configuration of the amino acid residues.

Experimental Section
General.TLC plates: Silicagel, Merck 60 Å, 254.Spots were visualized with UV light (254 nm and 366 nm) and with 1% ethanolic 4-(4-nitrobenzyl)pyridine (NBP).Melting points were determined using a Büchi apparatus, model 510.Analytical RP-HPLC was performed with a Waters 600S HPLC system (Waters 2489 UV/Vis detector, Waters 616 pump, Waters 717 plus autosampler, HPLC manager software from Chromax) using a C18 column (25 cm × 4.6 mm, 5 mm, Sigma) with a gradient of 0.1% TFA in H2O (A) and 0.08% TFA in MeCN (B), at a flow rate of 1 mL/min with UV detection at 220 nm, Rt in min.IR spectra were recorded as KBr pellets or film with a Bruker ALPHA spectrometer or a PerkinElmer Spectrum 100. 1 H and 13 C NMR spectra were recorded with a Bruker Avance DPX 250 (250 MHz) spectrometer, with chemical shifts (ppm) relative to TMS used as an internal standard.Multiplicities are marked as s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet.
Hydrolysis of peptides to amino acids hydrochlorides and derivatization for determination of enantiomeric purity by GC on ChirasilVal capillary column: Peptide (5 mg) in thick-walled test tube was treated with constant boiling hydrochloric acid (5 mL), freezed with dry-ice/ethanol, the pressure was reduced below 10 mbars and the tube was sealed-up.The sealed test tube was heated in boiling water for 24 h, then opened, and hydrochloric acid solution was evaporated to dryness.The residue was dissolved with distilled water (5 mL) and evaporated to dryness.Dissolution and evaporation were repeated three times.Solid residue was dried with P2O5 in vacuum desiccator, treated with methanol saturated with dry HCl for 24 h, evaporated to dryness, then dissolved in solution of trifluoroacetic acid anhydride (0.2 mL) in DCM (2 mL) for 12 h at room temperature.The solution was analyzed on a Chirasil-Val capillary column.Gas chromatography (GC) -HP 5840 II, FID (H2/air), split 1:30.

Table 4 .
Enantioselective synthesis of dipeptides 14a-g by coupling two equivalents of the racemic carboxylic