WO1996033162A1 - Compounds containing a substantially planar, fused ring system with at least 4 aromatic rings and their use as a chiral stationary phase in enantiomeric separation - Google Patents

Abstract

Compounds of the formula: Ar-L-(*)m in which Ar represents a substantially planar, fused ring system which contains at least 4 aromatic rings and which is of formula (I) wherein: n is 0 or 1; 0 means that the surrounding ring is aromatic when n is 1 and non-aromatic when n is 0; p is 0 or 1; and p is 0 when n is 0; R1 and R2, on the one hand, and R3 and R4, on the other hand, are chosen so that each forms a fused aromatic ring system together with the carbon atoms to which they are attached; R' is an alkyl group or hydrogen; and R5 is a group which, together with the atoms to which it is attached, forms one or more supplementary rings; L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom; and * is a radical with at least one chiral centre; and m is 1, 2 or 3; with the proviso that when * is a peptide or naturally occuring amino acid then the two chain atoms nearest the group Ar are carbon atoms. Kits for separating enantiomers comprising them; and processes for the separation of enantiomers using them are disclosed.

Description

COMPOUNDS CONTAINING A SUBSTANTIALLY PLANAR, FUSED RING SYSTEM WITH AT LEAST 4 AROMATIC RINGS AND THEIR USE AS A CHIRAL STATIONARY PHASE IN ENANTIOMERIC SEPARATION

The present invention relates to new compounds, and their use as a chiral stationary phase in separating

enantiomers. It relates particularly to chiral compounds facilitating the separation of enantiomers from a mixture of enantiomers during, or at the end of, a synthesis.

In organic synthesis of chiral molecules the

preferred procedure is to follow an enantiomerically

selective synthesis. However, such syntheses can be

difficult to design and involve a large number of steps.

The alternative procedure of following a non- enantiomerically selective synthesis followed by separation of enantiomers (either as intermediates or final products) has been little used due to the difficulty and expense of separating enantiomers. The present invention relates to the use of a chiral stationary phase (CSP) to effect such a separation.

Chiral stationary phases comprising a chiral selector immobilised on, for example, a support of silica gel are widely available (see W.H. Pirkle and T.C. Pochapsky, Chem. Rev. 1989, 89, 347 and Y. Dobashi and S. Hara, J. Org.

Chem., 1987, 52, 2490). However, they often fail to show adequate chiral selectivity. In addition they are often chemically unstable, and generally have low quantitative capacity.

Porous graphitised carbon (PGC) has recently been developed as a novel HPLC stationary phase. This

particulate porous graphite generally consists of not less than 98% carbon, comprising a plurality of particles,

preferably spherical, having a particle size range such that not more than 10% of the particles have sizes which are smaller than 50% of the number mean size and not more than 10% of the particles have sizes larger than 1.5 times the number mean size where said number means size lies

within the range from 1 to l000μm; the range from 1 to 20μm is suitable for high performance liquid chromatography, the range from 10 to l00μm is suitable for preparative liquid chromatography and the range from 50 to 1000 μm is suitable for industrial scale separation processing; said particles

(1) having a particle porosity within the range 40 to 80%; (2) having a specific surface area as measured by the

Brunauer, Emmet, Teller (BET) method with the range 5 to 500 m²/g, the preferred range being from 20 to 250 m²/g; (3) possessing the structure of either a two-dimensional graphite (otherwise known as a turbostratic carbon), or a three-dimensional graphite (of the Bernal type), such structures being identifiable by their X-ray powder

diffraction patterns obtained using CuK_α1,2 radiation

(wavelength 1.5418 A) as having, for two-dimensional graphite, a strong reflection at an angle 2θ=26.0° and for three-dimensional graphite, a corresponding strong

reflection at an angle 2θ=26.4°. A well known form of said two-dimensional graphite, is graphitised carbon black which has been used as a packing material in HPLC, but suffers from mechanical fragility and is generally unsuitable for the purpose of this invention. Recently other forms of 2- dimensional graphite, which have been specifically

developed for use in HPLC, have been disclosed: (a) porous graphites based upon silica templates, (I) GB-B-2035282,

(II) Czech patent CS-221197, (b) porous graphites made by heating carbon black mixed with a binder, EP-A-0484176 (c) porous graphites made by pyrolysis of spherical particles arising from the suspension polymerisation of a mixture containing an involatile oil, divinyl benzene, toluene and a catalyst EP-A-0 458 548. All of these latter forms of 2- dimensional graphite are suitable for the purpose of this invention, although the preferred forms are those described under (a) above.

PGC has a number of properties which make it

potentially a better support than silica for CSPs. These are:

1. High stereoselectivity since the surface of porous graphite is effectively flat; 2. Chemical inertness;

3. Uniform surface and high adsorptive

capacity which enhances substrate

loading.

Using these advantages resolutions of an enantiomeric mixtures have been achieved with dicyclohexyl tartrate as the chiral selector adsorbed on the PGC and also present in an aqueous mobile phase (E. Heldin, N.H. Huynh and C.

Pettersson, J. Chromatogr, 1992, 592, 339). However the ester hydrolyses with prolonged use.

Due to its inertness PGC cannot form covalent bonds. Consequently modification of the surface properties of graphite has to be achieved by the adsorption of suitable substances. Such modifier substances are preferably strongly adsorbed onto the surface of the graphite.

Compounds which can do this are described in GB-B-2251242. However, these compounds do not enable the separation of enantiomers. One of the aims, therefore, of the present invention is to provide a compound which adsorbs strongly to PGC and which contains a chiral selector able to

distinguish between enantiomers in a mixture.

The present invention provides a compound having the following formula:

Ar-L-(*)_m

in which Ar represents a substantially planar, fused ring system which contains at least 4 aromatic rings and which is of formula:

wherein:

n is 0 or 1;

0 means that the surrounding ring is aromatic when n is 1 and non-aromatic when n is 0;

p is 0 or 1; and p is 0 when n is 0;

R₁ and R₂, on the one hand, and R₃ and R₄, on the other hand, are chosen so that each forms a fused aromatic ring system together with the carbon atoms to which they are attached;

R' is an alkyl group or hydrogen; and

R₅ is a group which, together with the atoms to which it is attached, forms one or more supplementary rings;

L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom;

* is a radical with at least one chiral centre; and m is 1, 2 or 3;

with the proviso that the two chain atoms nearest the group Ar are carbon atoms in particular when * is a peptide or naturally occuring amino acid radical.

For the avoidance of doubt a phenyl group which is attached in the ortho position provides two chain atoms.

L preferably has from 2 to 10 chain atoms. More preferably L has 6 chain atoms.

The distance from the group Ar to the closest chiral centre is generally from 3 to 20 atoms. The distance is generally more than 7 atoms, preferably more than 8 atoms and generally less than 15 atoms.

Preferably all the chain atoms are carbon atoms.

Preferably the optional alkyl group substituents are C_1-4 alkyl. More preferably the chain is unsubstituted. The group Ar generally has at least 6 aromatic rings which are preferably hexagonal and preferably do not contain a heteroatom. Preferably the group Ar is

abbreviated as Tbf.

The radical * may be derived from a synthetic product or derived from a naturally occurring chiral product .

Typically it is a so called three point recognition type (see E. Eliel, Stereochemistry of organic compounds, J. Wiley (1994) pp 253-264) such as a Pirkle-type group or a naturally occurring product such as an antibiotic, for example, vancomycin, Pirkle-type groups preferably comprise a polar substituent, an electron-poor system, an aromatic system which is not poor in electrons and a chiral carbon centre bearing a hydrogen atom and/or an alkyl group.

Preferably the electron-poor system is aromatic. The polar substituent is preferably an amide which is preferably derived from an acid amine. The radical * may be chosen from other naturally occurring products, or derivatives thereof, known to the skilled man such as cyclodextrin derived radicals, carbohydrate derived radicals, alkaloid derived radicals, peptide derived radicals and steroid derived radicals. The radical * may be an ion pair. A suitable aromatic group is, for example, Tbf. Suitable supports are, for example, a silica gel or a graphite material. It is understood that when more than one radical * is present, these radicals may be the same or different. Thus the compounds of the present invention may comprise, for example, four chiral selectors.

Preferred are compounds of formula 1 :

in which n' is from 3 to 12, preferably 3 to 10;

R₆ is an alkyl, alkaryl, cycloalkyl, heterocyclic, aromatic or heteroaromatic group or an aryl group fused to a heterocycle, and is unsubstituted or substituted by one or more groups independently selected from:- NH₂,OH,OR,NR₂,NHR₂,OCOR,SR,SH and R;

and Ar₂ is an aromatic group which is unsubstituted or substituted by one or more groups independently selected from: - NO₂, CN, COOH, COOR, CONH₂, CONHR, CONR₂ , CHO, COR, SO₂R and SO₂OR;

wherein R is C₁-C₄ alkyl.

Preferably R₆ is an aromatic group or is derived from an amino acid and is optionally substituted by one or more groups independently selected from:- NH₂, OH, OR, NR₂, NHR₂, OCOR, SR, SH and R, in which R is as defined above.

Preferably Ar₂ is phenyl.

Preferably Ar₂ is substituted by one or more nitro groups .

Favoured natural products from which * is derived are typically derived are phenyl glycine, glycyl-L-proline, tartaric acid, glucose, cellulose and gluconic acid.

Specific compounds of the present invention include:

in which n' = 3, 6, 10.

The present invention also provides a graphite material, which is preferably graphite or activated charcoal, on which is adsorbed a compound of the present invention. There is also provided a process for preparing such a graphite material which comprises treating a graphite material with a compound of formula Ar-L-(*)m in which Ar and m are as defined above and L is a stable chain having at least two atoms in which the atoms are

independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or

heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom.

The present invention also provides a chamber filled with graphite material, which is preferably graphite or activated charcoal, on which is adsorbed a compound of the present invention. There is also provided a process for the preparation of such a chamber which comprises either packing a graphite material according to the present invention into a chamber or packing a graphite material into a chamber and then passing a solution of a compound of formula Ar-L-(*)m in which Ar, * and m are as defined above and L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom through the chamber.

The present invention also provides a kit for

separating enantiomers which comprises a compound of the present invention and a chamber filled with a graphite material, which is preferably graphite or activated

charcoal. The kit optionally further comprises a fraction- collecting assembly fitted with an ultraviolet (UV) or visible spectroscopic detector.

The present invention also provides a process for separating enantiomers which process comprises contacting a mixture of enantiomers with a compound of formula Ar-L-(*)m in which Ar, * and m are as defined above and L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or

heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom, simultaneously or successively, with a graphite material.

The compound of the present invention can be adsorbed onto the graphite material before or after being contacted with the mixture of enantiomers.

An enantiomer may be "bound" to the compound of the present invention. The enantiomer will thus be retained by the compound of the present invention when it is adsorbed on the graphite material. The nature of the bond is not clearly defined but it is likely to be intermediate between physical involving, for example, Van der Waals'

interactions, and chemical such as an ionic or non-covalent bond.

In a preferred process, the compound of the present invention is adsorbed on the graphite material and is then contacted with a mixture of enantiomers so that the

enantiomers elute out of the column at different rates.

More particularly the process comprises:

(A) (i) introducing a compound as described above in to a chamber filled with graphite material, which is preferably graphite or activated charcoal, such that the compound is adsorbed onto the graphite material;

(ii) introducing a mixture of enantiomers into the chamber such that either:

(a) one of the enantiomers is bound to the adsorbed compound such that it cannot be eluted while enantiomers which have not formed a bond with the adsorbed compound are eluted out of the column; or

(b) the enantiomers elute out of the column at different rates; and

(iii) in the case of (ii) (a) removing the compound bound to the enantiomer from the graphite material and recovering the bound enantiomer; or recovering the bound enantiomer from the adsorbed compound without

removing the compound from the graphite material; or

in the case of (ii) (b) collecting the separate fractions containing the different enantiomers.

In the case of (A) (ii) (a), an enantiomer not bound to the compound adsorbed on the graphite may be collected as it passes out of the chamber.

The present invention also provides a process for separating enantiomers, which comprises:

(B) (i) passing the mixture of enantiomers through a chamber filled with a graphite material, which is

preferably graphite or activated charcoal, in the presence of a compound of the present invention such that the compound is bound to the enantiomer and adsorbed on the graphite material and such that enantiomers not bound to the compound of the present invention elute out of the column; and either

(ii) (a) removing the compound bound to the enantiomer from the graphite material and recovering the bound enantiomer; or

(b) recovering the bound enantiomer from the adsorbed compound without removing the compound from the graphite material.

In either case an enantiomer not bound to the compound adsorbed on the graphite may be collected as it passes out of the chamber. In this embodiment the compound of the present invention and the mixture of enantiomers can be reacted together before passing the enantiomers through the chamber or, alternatively, the compound and the mixture can be applied separately to the chamber.

The two general approaches to the loading of the compounds of the present invention onto the PGC-material, can be described as 1) External loading and 2) Dynamic loading.

1) External loading involves the treatment of loose graphite material with the required amount of TBF- derivative which is generally dissolved in a suitable solvent system in which the latter is sparingly soluble. The. coated material is then packed into a column.

Examples of solvent systems are pure acetonitrile and a mixture of dichloromethane, acetonitrile and methanol generally in a volume ratio of 2:10:35.

2) Dynamic loading is a method in which a solution of the TBF-derivative is pumped through a packed column thereby coating it. The column is assumed to have a single monolayer when material is observed to be eluting from the end of the column i.e. when breakthrough has occurred.

An example of a solvent system for dynamic loading experiments is acetonitrile in which the TBF-derivative is only sparingly soluble.

The chiral stationary phases of the present invention have a number of advantages over those currently available.

1. It is possible to coat and strip the phase from a packed column without the need for specialised equipment.

2. a) A coated graphite material when stripped regains all of the properties of the original graphite material and can be used for standard separations or it can be recoated.

b) The stripped chiral coating can be collected and purified to the original quality.

Thus even a badly contaminated column can be fully regenerated.

3. Any chamber can be coated, stripped and

recoated with any number of different chiral selectors which means that one column can be used for a range of different chiral phases.

4. The selectivities found with graphite material chiral stationary phases and silica material chiral

stationary phases are significantly different although overall they are of similar magnitudes.

The above advantages indicate that the chiral

stationary phases of the present invention have far- reaching advantages for preparative and semi-preparative scale work where material costs are large.

Groups of formula I:

in which n, O, P, R₁, R₂, R₃, R₄, R₅ and R' are as defined above can be derived by known methods from

compounds prepared by methods and techniques well-known in the art, see GB-B-2251242 for example. In particular, compounds of formulae 12a and 12b:

can be prepared by known methods e.g. Brown et al.,

Tetrahedron Lett., 34, 7129-7132, (1993). Generally they can be deprotonated using hydroxide ions to give a group of formula 13:

In general, known methods can be used to graft one end of a linker compound onto the group Ar. This linker compound corresponds tc the group L except that it has two groups suitable for grafting on each end. The portion of the linker compound grafted onto Ar corresponds to L. This grafting can be followed or preceded by reaction between a suitable group on the other end of the linker compound with a chiral molecule corresponding to radical *. In the formula Ar-L-(*)_m, * is understood to include any residue from the group in the linker compound which has undergone reaction with the chiral molecule corresponding to * and which does not fall within the above definition of L.

In particular, the following processes are preferred to make compounds of the present invention, together with the processes exemplified in the Examples.

Tbf (tetrabenzofluorene) compounds of formula 2:

can be prepared as shown in Scheme 1, They are produced via a compound of formula 14 :

in which X is a group which can easily be transformed into an amine. Compounds of formula 14 in which X is N(BOC)₂ (ie compounds of formula 16) can be produced by the reaction between compounds of formulae 13 and 15 as shown in Scheme 1.

Compounds of formula 14 in which X = CO₂H, Br and OH have also been prepared by known methods.

reflux, iv) C₇H₈O₃S.H₂O, DCM, reflux, v) NaOH, H₂O, DCM; vi) R N-13, 5-dinitro benzoyl-oi-phenylglycine, EEDQ, THF.

Compounds of formula 15 can be produced from

commercially available dibromo precursors as shown.

Compounds of formula 13 are produced by reacting compounds of formula 12b with 40% aqueous tetra-n-butyl ammonium hydroxide.

Compounds of formula 16 are then transformed into the amine p-toluenesulfonate salt from which the free amine can be liberated for reaction with R (N-3, 5-dinitrobenzoyl) -α- phenylglycine to give the desired compounds of formula 2 using 2-ethoxy-1-ethoxy-carbonyl-1,2-dihydroquinoline

(EEDQ) as coupling agent.

An alternative route for the preparation of compounds of formula 17 is shown in Scheme 2:

Reagents and Conditions: i) (BOC)₂O, THF, ii) I₂, PPh₃, imidazole, Et₂O, CH₃CN, 0°C, iii) 13, 1,4-dioxane, reflux, iv) C₇H₈O₃S.H₂O, DCM, reflux.

This route does not require chromatographic

purification of synthetic intermediates.

The starting materials for scheme 2 are all commercially available.

Compounds of formula 15 in which X is CO₂H (i.e. the compound of formula 23) can be produced by a reaction between compounds of formulae 13 and 21 followed by deprotection as shown in Scheme 3.

The starting materials for Scheme 3 are all

commercially available.

The compound of formula 23 was converted into its N- hydroxy succinimide active ester and reacted with

vancomycin to give the desired compound 11.

Compounds having more than one chiral centre are typically produced as shown in reaction Scheme 4.

Reagents and conditions: i) ClCO₂Et, Et₃N, THF, -5°C; ii) NaOH, H₂O, 1,4 dioxane; iii) 17 (as free amine),

ClCO₂Et, Et₃N, THF, -5°C; iv) TFA/DCM; v) MeOH, DCM, 2MNaOH (aq) vi) R-(N-3,5, dinitrobenzoyl)-α-phenylglycine.

The starting materials for Scheme 4 are all

commercially available.

Alternative ways of producing compounds having more than one chiral centre are shown in reaction Schemes 5 and 6. The first step in Scheme 5 is as described in Brunson and Riener, J. Am. Chem. Soc. 1943, 65, 23. Again, all starting materials for both schemes are commercially available.

The compound resulting from scheme 4 can be reacted with a compound of formula :

Ar-L-COCl

in which Ar and L are as defined for the compounds of the present invention, to give the desired product.

Other di- or tri-nitrile precursors to compounds of formula Ar-L-(*)_m can be made by methods known in the art, for example, as shown in schemes 7 and 8. All starting materials in these schemes are commercially available.

The compounds of the present invention are generally loaded onto PGC by adding them in a solvent. They can be removed by washing with a stronger solvent or by boiling simultaneously or successively in one or more solvents, for example by boiling 1,4-dioxane followed by boiling in toluene. The PGC can then be reused either with the same or a different compound of the present invention.

The following section illustrates typical loading and removal procedures for the compounds of the present

invention onto PGC:

Loading

Solutions of known concentrations of a Tbf compound in acetonitrile were added to loose PGC and also to a prepacked PGC column, with monitoring for breakthrough at 365 nm and 254 nm respectively. The amounts adsorbed determined using these methods were ca 4.5xl0^-5 mol g^-1 and 5.5xl0^-5 mol g^-1, respectively. The values correspond approximately to monolayer coverage. With these results in hand, CSPs were prepared by adsorbing amides 2 onto prepacked columns or onto loose PGC prior to packing. Both methods worked well although the latter is more convenient due to the low solubility of amides 2 in acetonitrile and the latter method was used in the preparation of the CSPs whose results are reported herein. In general preparation of a CSP involved dissolving the required amide 2 in DCM (2ml), CH₃CN (10ml) and MeOH (35ml). PGC (1.1g) was then added and the mixture was allowed to stand with occasional stirring for 1 hour whereupon the solvent was removed by pipette and the PGC was washed with MeOH (3x30ml) then packed into a 4.6x100mm column.

A column coated in amide 2 was washed through with toluene, with the first 200 ml washing off 13mg/73% of the amide 2. Washing through with more toluene failed to remove any of the amide 2. A test chromatogram confirmed that not all the amide 2 had been washed off. The sample washed off this column was reabsorbed onto PGC. This column was able to resolve a test sample of 9-anthrylphenyl alcohol.

A column coated in amide 2 was dismantled and the PGC boiled in 1, 4 dioxane. The weight of the recovered sample was less than had been adsorbed. The PGC was boiled in toluene, which removed all the remaining amide 2. The PGC was repacked into a column and a test chromatogram showed it behaved as 'fresh' PGC.

The material recovered by boiling the PGC in dioxane and toluene was also adsorbed onto PGC and repacked into a column. In this case it was not possible to achieve chiral resolution of the p-anthryl phenyl alcohol. Tbf

hexanoxycarboxylglycyl-L-proline (3) is not washed off PGC by aqueous sodium hydroxide, but is washed off by

triethylamine.

The following Examples further illustrate the present invention.

General Methods and Equipment. All solvents applied as , reaction media were HPLC or AR grade. Unless otherwise stated, materials were obtained from commercial suppliers and used without further purification. M.p.s were

determined on a Buchi 510 melting point apparatus and are uncorrected. TLC analyses were carried out on Merck

Kieselgel 60F₂₅₄ aluminium backed plates (0.2mm).

Visualisation of spots was accomplished with UV light, by dipping in either an ethanolic solution of p-anisaldehyde, which also contained sulfuric and acetic acid, or aqueous potassium permanganate, followed by heating. Crude products were purified by dry flash chromatography using Merck Kieselgel 60 H. Elemental analyses (CHN) were obtained using a Perkin-Elmer 2400 CHN Elemental Analyzer. Optical rotations were measured on an Optical Activity AA1000 polarimeter, and are given in 10^-1 deg cm² g^-1. UV spectra were recorded on a Varian Cary 210 from 400-235nm and IR on a BIO-RAD FTS-7 machine. NMR spectra were recorded using a Bruker AC 250 at 250MHz for ¹H and 68.9 MHz for ¹³C, unless indicated otherwise. Chemical shifts are reported in parts per million (δ) and J values are given in Hz. For ¹³C NME 1°, 2°, 3° and 4° denote methyl, methylene, methine and quaternary carbon respectively.

Mass spectra were obtained on a Kratos MS 50 TC mass spectrometer using fast atom bombardment (FAB). HPLC equipment combined a Gilson 305 and 302 pump, rheodyne Laboratory Data Control (LDC) 7125 injector valve, LDC 1204A detector, Hewlett Packard 3390A integrator and a Kipp & Zonen recorder. Stainless steel columns 100 mm long and 4.6 mm bore were packed with 7 micron Hypercarbº by Shandon HPLC.

Example 1

Preparation of N-(3,5 Dinitrobenzoyl)-α-phenylglycyl 3-(17- tetrabenzo [a , c,g, i] fluorenyl)propyl amide 2a

Preparation of Di -tert -butyl N- ( 3 -bromopropyl )

aminodicarboxylate 15a Di-tert-butyl iminodicarboxylate (969mg, 4.46 mmol) was dissolved in THF (20 cm³) and DMF (20ml) under an atmosphere of nitrogen. Sodium hydride (137 mg of an 80% dispersion in oil, 4.57 mmol) was added and the mixture heated at 65°C for 2.5 hours. 1,3-Dibromopropane (2.00 cm³, 19.7 mmol) was added and the mixture heated at 65°C for 3 hours. On cooling to room temperature, ether (30cm³) and water (30 cm³) were added. The separated organic layer was washed with water (2x30 cm³) and brine (20 cm³). It was dried with MgSO₄ and filtered. Removal of solvent at reduced pressure gave a brown oil. The excess dibromide was distilled from this oil at 0.8 mmHg. The residue after distillation was dry flash chromatographed eluting

initially with 2% ether/hexane. This gave 15a as a

colourless oil (880mg, 58%) ;_v_max (neat)/cm^-1 2980, 2935 (CH), 1791, 1744, 1694 (C=O) ;_δ_H (250 MHz;

CDCl₃)3.58(2H,t, J6), 3.26 (2H.t,J6.5), 2.00 (2H, quin, J7), 1.37(18H,S,C(CH₃)₃);_δ_c(62.9 MHz ; CDCl₃) 152.05 (2x4°, C=O), 82.11(2x4°, C(CH₃)₃)), 44.86(2°), 31.83(2°), 30.11(2°), 27.73 (6x1°, C(CH₃)₃) ) ;m/z (FAB) 338 (MH⁺,30%) , 282(75), 222 (85), 146 (50), 56(100).

Preparation of Di-tert-butyl N-(3-(17- tetrabenzo [a,c,g,i]fluorenyl)propyl) aminodicarboxylate 16a Tbf 12b (286 mg, 0.78 mmol) was dissolved in

refluxing 1,4-dioxane (12 cm³) under an atmosphere of nitrogen. 40% aqueous ⁿBu₄NOH (0.5cm³, 0.75 mmol) in 1,4- dioxane (1.5 cm³) was added and the mixture refluxed for 5 minutes. After cooling the yellow ammonium salt 13 was filtered off under nitrogen and washed with 1,4-dioxane

(2x) and ether (2x). It was used in the next stage without further purification.

The ammonium salt 13 and di-tert-butyl N-(3- bromopropyl) aminodicarboxylate 15a (300 mg 0.89 mmol) were mixed in 1,4-dioxane (15 cm³) under nitrogen and the mixture refluxed for a further 14 hours. On cooling to room temperature the mixture was filtered and the solvent removed at reduced pressure. Ether was added and the resulting precipitate filtered off, washed with hot ether and discarded. The ether was removed at reduced pressure to give a red oil. This was dry flash chromatographed using Kieselgel H, eluting initially with 50% DCM/hexane with 10% increments. This gave the product 16 mixed with an unidentified product. The mixture was dry flash chromatographed using Kieselgel H, eluting initially with 20% ether/hexane. This gave the di BOC compound 16a as a yellow oily solid. The di-BOC compound was recrystallised from ether (-15°C) overnight as a light brown solid (190 mg, 39%), m.p. 190-193 °C (Found: N, 2.48. C₄₂H₄₁NO₄ requires N, 2.25%);_λ_max (DCM)/nm 382 (e/dm³ mol^-1 cm^-1, 17686), 365 (18256), 301 (44487), 289 (36526), 278 (sh), 260 (67433), 254 (74925), 240 (56662) ;_v_max (DCM mull)/cm^-1 2978, 2918 (CH), 1778, 1742 (C=O);_δ_H (250 MHz; CDCl₃)8.78 (4H, dm, J8), 8.67 (2H, dd, J8 and 2), 8.23-8.12 (2H, m), 7.72-7.41 (8H,m), 5.04 (1H, t, J4), 3.06 (2H, t, J 7.5), 2.62-2.54 (2H, br.m), 1.43 (18H, s, C(CH₃)₃), 0.67-0.46 (2H, br.m);- _δ_c(62.9 MHz; CDCl₃) 151.77 (2 X 4°, C=O), 143.59 (2 x 4°), 136.89 (2 X 4°), 131.34 (2 x 4°), 130.47 (2 X 4°), 128.57 (2 X 4°), 127.92 (2 x 4°), 127.47 (2 x 3°), 126.89 (2 X 3°), 125.95 (2 X 3°), 125.09(2 X 3°), 124.20 (2 x 3°), 123.49 (4 X 3°), 81.48 (2 X 4°, C(CH₃)₃)), 46.47 (3°), 45.94(2°), 30.62 (2°), 27.43 (1°, C (CH₃) ₃) ), 21.91 (2°); m/z (FAB) 623 (M⁺, 38%), 468 (62), 365 (44), 325 (100), (Found: M⁺, 623.30353. C₄₂H₄₁NO₄ requires 623.30354).

Preparation of 3-(17- Tetrabenzo [a,c,g,i]fluorenyl)propylamine p-toluenesulfonic acid salt 17a

Di-tert-butyl N-(3-(17- tetrabenzo [a, c, g, i]fluorenyl)propyl) aminodicarboxylate 16a (192 mg, 0.31 mmol) and p-toluenesulfonic acid monohydrate (67 mg, 0.35 mmol) were dissolved in DCM (20 cm³) under an atmosphere of nitrogen. The mixture was refluxed for 20 hours. On cooling solvent was removed at reduced pressure. DCM (2 cm³) and ether (8 cm³) were added to the white solid and the mixture centrifuged. The solvent was decanted off. The above was repeated until the solvent was no longer red (3x). The solid was dried. The amine salt 17a was

obtained as a white solid (172 mg, 94%), m.p. 156-159°C (Found: C, 77.99; H, 5.74; N, 2.47. C₃₉H₃₃NO₃S requires C, 78.63; H, 5.58; N, 2.35%);_λ_max (DCM/MeOH (l:l))/nm 380 (e/dm³ mol^-1 cm^-1, 18263), 365 (19199), 301 (44487), 289 (36526), 278 (sh), 260 (67433), 254 (74925), 240

(56662) ;_v_max (bromoform mull)/cm^-1 3457, 3200-2900 (NH), 1608, 1500;_8δ_H (360 MHz; d₆-DMSO) 8.93 (4H, t, J8), 8.56 (2H, d, J8), 8.36 (2H, d, J8 ), 7.81-7.70 (8H, m), 7.50 (2H, d, J8), 7.24 (3H, br.s, NH₃), 7.11 (2H, d, J8), 5.36 (1H, t, J4), 2.65-2.60 (2H, br.m), 2.28-2.20 (5H, br.m), 0.46- 0.46 (2H, br.m) ;_δ_c (50.3 MHz; d₆-DMSO) 145.62 (4°), 144.02 (2x4°), 137.93 (4°), 136.03 (2x4°), 131.10 (2x4°), 130.08 (2x4°), 128.26 (2x3°), 128.22 (2x4°), 127.59(2x3°), 127.33 (2x3°), 126.86(2x3°), 126.78 (2x4°), 126.35 (2x3°), 125.66 (4x3°), 124.83 (2x3°), 124.22 (2x3°), 123.95 (2x3°), 46.09 (3°), 38.76 (2°), 30.51 (2°), 20.94 (1°), 20.63 (2°); m/z (FAB) (595 M⁺, 0.5%), 424 (100, M⁺ -C₇H₇SO₃), 365 (47)

(Found: M⁺ -C₇H₇SO₃, 424.20655. C₃₂H₂₆N requires 424.20651).

Preparation of N-(3, 5 Dinitrobenzoyl)-a-phenylglycyl 3- (17-tetrabenzo[a,c,g,i]fluorenyl)propyl amide 2a

The amine salt 17a (146 mg, 0.25 mmol) was dissolved in methanol (4 cm³) and DCM (20 cm³). This mixture was then washed with 0.4M NaOH aq. (5 cm³), water (10 cm³) and brine (5 cm³). It was dried with MgSO₄ and filtered.

Removal of solvent gave the free amine as a foam, which was used in the next stage of the reaction without further purification.

The free amine was dissolved in THF (5 cm³) under an atmosphere of nitrogen R-N-(3,5- Dinitrobenzoyl)phenylglycine (93 mg, 0.27 mmol) was added followed by EEDQ (66 mg, 0.27 mmol). The mixture was stirred at room temperature for 18 hours. Solvent was removed at reduced pressure to give an orange solid. This was dissolved in DCM (2 cm³) and hexane (10 cm³) was added. The resulting orange precipitate was filtered off and washed with hexane. The above was repeated. The orange solid was absorbed onto silica and dry flash

chromatographed using Kieselgel H, eluting initially with DCM then 0.2% MeOH/DCM up to 6%. This gave the amide 2a as an orange solid (131 mg, 71%) , m.p. 167-170°C (Found: C, 75.22; H, 4.80; N, 7.56. C₄₇H₃₄N₄O₆ requires C, 75.19; H, 4.57; N, 7.46%); [α] _D at 24°C = -51.1 (c=4.85xl0^-3 in

CC1 ₃); -λ_max (DCM)/nm 382 (e/dm³ mol^-1 cm^-1, 14489), 365

(15341), 301 (33239), 289 (29403), 260 (sh), 254

(72869) ; _v_mx (DCM mull) /cm^-1 3394, 3301, 1644 (C=O),

1540;_δ_H (200 MHz; CDCl₃) 8.9 (1H, t, J2), 8.73-8.58 (8H, m), 8.29 (1H, d, J6.5), 8.18-8.09 (2H, m), 7.68-7.59 (8H, m), 7.19-7.04 (5H, m), 5.09 (1H, d, J6.5), 4.98 (1H, t, J4), 4.88 (1H, t, J6), 2.85-2.74 (1H, m), 2.55-2.40 (3H, br.m), 0.46-0.39 (2H, br.m) ;_δ_c (90.6 MHz; CDCl₃)

169.35(4°), 160.50 (4o), 147.29 (2x4°), 143.28 (4°), 142.94 (4°), 136.83(4°), 136.44(4°), 136.25 (4°), 135.43 (4°), 130.59 (2x4°), 129.76 (4°), 128.58 (2x3°), 128.24 (3°), 127.92(2x4°), 127.32 (2x4°), 127.23 (4x3°), 127.05 (3°), 126.77 (3°), 126.64 (3°), 126.23 (2x3°), 125.86 (3°),

125.76 (3°), 125.61 (3°), 124.93 (2x3°), 123.89 (2x3°), 122.96 (4x3°), 120.17 (3°), 56.95 (3°), 45.91 (3°), 39.30 (2°), 30.10 (2°), 21.79 (2°),; m/z (FAB) 751 (MH⁺, 72%), 750. (M⁺, 40), 433 (100), 365 (62) (Found: MH⁺, 751.25565. C₄₇H₃₅N₄O₆ requires 751.25564). Example 2

Preparation of N-(3,5 Dinitrobenzoyl)-α-phenylglycyl 6-(17- tetrabenzo [a,c,g,i]fluorenyl)hexyl amide 2b

Method A to prepare 6-(17-Tetrabenzo [a,e,g,g]fluorenyl hexylamine p-toluene sulfonic acid 17b

Preparation of Di-tert-butyl N-(6-bromohexyl)

aminodicarboxylate 15b

This compound was obtained by the above procedure using di-tert-butyl iminodicarboxylate (20.64 g, 95.1 mmol), sodium hydride (3.30 g of an 80% dispersion in oil, 0.11 mol), 1,6-dibromohexane (78.85 g, 0.32 mol), THF (130 cm³) and DMF (130 cm³). After distillation the crude reaction mixture was dry flash chromatographed eluting initially with 4% ether/hexane. This gave 15b as a colourless oil (14.82 g, 40%);_V_{ma x} (neat)/cm^-1 2980, 2930, 2865(CH), 1780, 1746, 1694 (C=O);_δ_H (250 MHz ; CDCl₃)

3.52(2H,t, J7), 3.36 (2H,t,J7), 1.82 (2H, q, J7), 1.57-1.24 (24H, m, includes at 1.46 (18H, s, C (CH₃) ₃) ) ;_δ_c (62.9 MHz CDCl₃) 152.24 (2x4°, C=O), 81.53 (2x4°, C(CH₃)₃), 45.80(2°, CH₂N), 33.19 (2°), 32.28 (2°), 27.66 (6x1°, C(CH₃)₃), 27.42 (2°), 25.53 (2°), 22.39(2°); m/z (FAB) 382 (MH⁺, 11.5%), 380 (MH⁺, 22), 326 (18), 324 (32), 270 (100), 268 (100), 56 (100) (Found: MH⁺, 380.14369. C₁₆H₃₁ ⁷⁹BrNO₄ requires 380. 14368. Found: MH⁺, 382.14174. C₁₆H₃₁ ⁸¹BrNO₄ requires

382.14171).

Preparation of Di-tert-butyl N-(6-(17- tetrabenzo [a,c,g,i]fluorenyl)hexyl) aminodicarboxylate 16b This compound was obtained by the above procedure using Tbf 1(2.19 g, 5.98 mmol) in 1, 4-dioxane (50 cm³), 40% aqueous ⁿBu₄NOH(4.0 cm³, 6.0 mmol) in 1, 4-dioxane (12 cm³) and di-tert-butyl N- (6-bromohexyl) aminodicarboxylate 15b in 1, 4-dioxane (120 cm³). The reaction was complete after 2 hours at reflux. The crude reaction mixture was dry. flash chromatographed eluting initially with 50%

DCM/hexane. This gave product 16b as a pale yellow solid (1.48 g, 37%), m.p. 164-167°C (Found: N, 2.23. C₄₅H₄₇NO₄ requires N, 2.10%) ; _λ_max (DCM) /nm 382 (e/dm³ mol^-1 cm^-1,

17829), 365 (18312), 302 (41442), 290 (34696), 280 (32286), 262 (62645), 254 (68428), 240 (51079) ;_v_max (DCM mull)/cm^-1 2980, 2930, 2860 (CH), 1776, 1738, 1692 (C=O);_δ_H (250 MHz; CDCl₃) 8.79 (4H, m), 8.67 (2H, dd, J8 and 1), 8.22 (2H, m), 7.75-7.6 (8H, m), 4.97 (1H, t, J 4.5), 3.20 (2H, t, J7,

CH₂N) , 2 . 58 ( 2H, m) , 1 . 35 ( 18H , s , C ( CH₃) ₃) ) , 1 . 13 ( 2H, m) . 0 . 77 ( 4H, m) , 0 . 35 ( 2H, m) ; δ_c ( 62 . 9 MHz ; CDCl₃) 152 . 21 ( 2 x 4°, C=O) , 144.01 (2x4°) , 136.45 (2x4°) , 131.02 (2x4°) , 130.12 (2x4°) , 128.48(2x4°) , 127.75(2x4°) , 127.19(2x3°) , 126.51(2x3°) , 125.51(2x3°) , 125.38(2x3°) , 124.74(2x3°) , 124.21(2x3°) , 123.28 (2x3°) , 123.24(2x3°) , 81.50 (2x4°, C(CH₃)₃) , 46.73(3°) , 45.88(2°, CH₂N) , 29.05 (2°) , 28.55 (2°) , 27.73 (6x1°, C(CH₃)₃) , 25.99 (2°) , 22.22 (2°) ;m/z (FAB) 665(M⁺, 46%) , 510 (18.7) , 466 (8) , 365 (42) , 56 (100) (Found: M\ 665.35046. C_4SH₄₇NO₄ requires 665.35049) . Preparation of 6- (17-

Tetrabenzo[a,c,g,i]fluorenyl)hexylamine p toluenesulfonic acid salt 17b

From 16b. This compound was obtained by the above procedure using di-tert-butyl N-(6-(17- tetrabenzo[a,c,g,i]fluorenyl)hexyl) aminodicarboxylate 16b

(286 mg, 0.43 mmol), p-toluenesulfonic acid monohydrate (83 mg, 0.44 mmol) and DCM (25 cm³). 17b was isolated as a white solid (277 mg 98%), m.p. 153-155°C. Method B to prepare 18b

Preparation of tert-Butyl N-(6-hydroxyhexyl)

aminocarboxylate 18b

6-Aminohexan-1-ol (24.93 g, 0.21 mol), was suspended in THF (170 cm³) under nitrogen. Di-tert-butyl dicarbonate (44.11 g, 0.20 mol), was added to the suspension

portionwise at room temperature. After the final addition the. mixture was stirred for 1 hour. The mixture was filtered and the volume of THF reduced. Ether (150 cm³) was added and the mixture washed with 2M HCl aq. (2x50cm³), water (50cm³) and brine (25cm³). It was dried with MgSO₄ and filtered. Removal of solvent at reduced pressure gave an oil. This was dried at 0.8mmHg. On cooling in a refrigerator 18b was obtained as a white solid (42.10 g, 96%), m.p. 35-36.5 °C (Found C, 60.37; H, 10.99; N, 6.14. C₁₁H₂₃NO₃ requires C, 60.80; H, 10.67; N, 6.45%); v_{ma x}

(neat)/cm^-1 3355 (OH, NH), 1693 (C=O);_δ_H (250 MHz; CDCl₃) 4.58 (1H, br.s, NH), 3.58 (2H, t, J6.5, CH₂OH), 3.07 (2H, q, J6.5, CH₂N) , 1.97 (1H, s, exchanges with D₂O, OH) , 1.55- 1.26 (17H, m, includes at 1.40, 9H, s, C (CH₃) ₃) ;_δ_c (62.9 MHz; CDCl₃) 155.99 (4°, C=O) , 78.70 (4°, C(CH₃)₃) , 61.95 (2°, CH₂OH) , 40.00 (2°, CH₂N) , 32.23 (2°) , 29.71(2°) , 28.14 (3x1°, C(CH₃)₃) ) , 26.19 (2°) , 25.11 (2°) ; m/z (FAB) 218 (MH⁺, 48%) , 217 (M⁺, 3.3) , 162 (100) , 154 (33.3) , 144

(23.2) , 138 (15.5) , 137 (39) , 136 (24.1) . (Found: MH\ 218.17531. C₁₁H₂₄NO₃ requires 218.17562) . Preparation of tert-Butyl N-(6-iodohexyl) aminocarboxylate 19b

tert-Butyl N-(6-hydroxyhexyl) aminocarboxylate 18b

(27.56 g, 0.127 mol), triphenylphosphine (40.30 g, 0.154 mol) and imidazole (12.63 g, 0.186mol) were dissolved in ether/acetonitrile (3:1) (1000 cm³) with mechanical

stirring under an atmosphere of nitrogen. This mixture was cooled to 0°C with an ice/water bath. Iodine (41.94 g, 0.165 mol) was added and the mixture stirred at 0°C for 1 hour. Water (900 cm³) was added. The separated organic layer was washed with water (900 ml), sat^d Na₂S₂O₃ aqueous, (250 cm₃), water (500 cm³) and brine (200 cm³). It was dried with Na₂SO₄ and filtered. Solvent was removed at reduced pressure to give an oil and white solid. Hexane (5x50 cm³) was added and decanted off the white solid. The hexane was removed to give a yellow oil. This was filtered through silica using 10% ether/hexane. This gave 19b as a colourless oil. It was dried at 0.8 mmHg and solidified as a white solid in the refrigerator (37.03 g, 89%), m.p. 24- 25°C (Found: C, 40.25; H, 7.16; N, 4.25. C₁₁H₂₂INO₂ requires C, 40.37; H, 6.78; N, 4.28%); _v_max (neatj/cm^-1 3354 (NH), 1696 (C=O) ;_δ_H(250 MHz;CDCl₃) 4.52 (1H, br.s, NH), 3.15 (2H, t, J7, CH₂I), 3.07 (2H, q, J6.5, CH₂N), 1.81 (2H, m), 1.51-1.25(15H, m, includes at 1.41 9H, s, (C (CH₃) ₃) ;_δ_c (62.9 MHz; CDCl₃) 155.78 (4°, C=O), 78.71 (4°, C(CH₃)₃), 40.28(2°, CH₂N), 33.13 (2°), 29.91 (2°), 29.68 (2°), 28.24 (3x1°,

C(CH)₃)₃), 25.44 (2°), 6.83 (2°, CH₂I); m/z (FAB) 328 (M⁺, 13.7%), 326 (10.5), 273 (37.5), 272 (100), 270 (25), 228 (53), 226 (35.4), 145 (17.5), 144 (44.4), 137 (22.5), 136 (11.5) (Found: MH⁺, 328.07624. C₁₁-I NO₂ requires

328.07735).

From 12b using 19b as the alkylating agent. The ammonium salt 13 was prepared as above using Tbf 12b (20.24 g, 55.30 mmol), degassed 1,4-dioxane (500 cm³) and 40% aqueous ⁿBu₄NOH (36.90 cm³, 55.35 mmol), degassed 1,-4- dioxane (100 cm³)

The iodide 19b (18.06 g, 55.23 mmol) in warm degassed 1,4-dioxane (250 cm³) was added to the ammonium salt 13 prepared above under an atmosphere of nitrogen. The mixture was then brought to reflux and the mixture refluxed for 5 minutes after all the ammonium salt dissolves. Total time at reflux was 12 minutes. The mixture was then stirred in an ice/water bath until precipitation of ⁿBu₄NI was complete and then at room temperature for 5 minutes. The precipitate was filtered off and washed with ether.

The solvent of the filtrate was then removed at reduced pressured to give a red oil. This was triturated with hexane (4x50 cm³) and then filtered through silica with 50% DCM/hexane. After removal of solvent and drying at 0.8 mmHg a foam was obtained 32.06 g > 100%. A small amount of this material was purified by chromatography for

characterisation purposes. The major component isolated was the mono BOC 20b compound as a yellow solid m.p. 69-

72°C (Found: C, 79.57; H, 6.97; N, 2.38. C₄₀H₃₉NO₂ requires C, 84.96; H, 6.95; N, 2.48%) ;_λ_max (DCM) /nm 382 (e/dm³ mol^-1 cm^-1, 17152), 365 (17656), 301 (45401), 288 (43384), 276 (sh), 262 (72138), 254 (756670) ;_v_max (DCM mull)/cm^-1 3436, 3355 (NH), 1711 (C=O) ;_δ_H (250 MHz; CDCl₃) 8.80 (4H, dt, J8 and 2), 8.67 (2H, dd, J8 and 1.5), 8.23 (2K, m), 7.72-7.58 (8H, m), 5.00 (1H, t, J4.4), 3.70 (1H, br.s, NH), 2.65 (2H, q, J6.5), 2.61-2.55 (2H, m), 1.36 (9H, s, C(CH₃)₃), 0.95- 0.85 (2H, m), 0.8-0.65 (4H, m), 0.4-0.25 (2H, m);_δ_c (6219 MHz; CDCl₃) 155.21 (4°, C=O), 144.07 (2x4°), 136.60 (2x4°), 131.20(2x4°), 130.22 (2x4°), 128.59 (2x4°), 127.86 (2x4o), 127.30 (2x3°), 126.63 (2x3°), 125.74 (2x3°), 125.50 (2x3°), 124.87 (2x3°), 124.29 (2x3°), 123.37 (2x3°), 123.33 (2x3°), 78.62 (4°, C(CH₃)₃), 46.70 (3°), 40.07 (2°, CH₂N) , 33.14 (2°), 29.32 (2o), 28.77 (2o), 28.27 (3x1°, C(CH₃)₃), 25.70 (2°), 21.81 (2°),; m/z (FAB) 565 (M\ 100%), 510 (43.6), 466 (28.4), 366 (60.8), 365 (85.3), 364 (87.0), 363 (82.5), 58 (86.6), 56 (40.9) (Found: M⁺, 565.29883. C₄₀H₃₉NO₂ requires 565.29808). The brown foam from above (31.76 g) was dissolved in DCM (250 cm³) under nitrogen, p- Toluenesulfonic acid monohydrate (8.5g, 0.8eq.) was added and the mixture refluxed with stirring for 18 hours. The mixture was then cooled in an ice salt bath and the off white solid filtered off from the red solution. The solid was dissolved in methanol (125 cm³), filtered if necessary, and then removed at reduced pressure to give a light brown solid. DCM (60 cm³) was added and the solution allowed to stand. The resulting white precipitate was filtered off and washed with DCM (2x). The DCM was removed and DCM (30 cm³) was added to the solid. The resulting white solid was filtered off and combined with the first crop. The

combined first and second crops were dried at 0.8 mmHg for

3 hours. This gave the amine salt 17b as a white solid (20.48 g, 58%) mp 152-155°C. A third crop was obtained (1.52 g) mp 145-149°C.

The initial red filtrate was reduced in volume (50 cm³) and p-toluenesulfonic acid monohydrate (1.5 g) was added. The mixture was refluxed under nitrogen for 5 hours and. cooled in an ice/water bath. The red precipitate was filtered off and washed with DCM (3x) to give a pink solid. This was dissolved in hot methanol (25 cm³). The methanol was removed at reduced pressure and DCM (10 cm³) was added to the solid and the solution allowed to stand for 1 hour. DCM (20 cm³) was added to the white solid. It was then filtered off and washed with DCM. After drying (0.8 mmHg) the white solid (2.40 g) melted at 152-155°C.

Total amount of amine salt 17b prepared from Tbf 12b (20.24 g, 55.30 mmol) was 22.72 g 64% m.p. 152-155°C

(Found: C, 79.08; H, 6.62; N, 2.29. C₄₂H₃₉NO₃S requires C, 79.09; H, 6.16; N, 2 . 20% ) . _λ_max (DCM:MeOH 1:1) /nm 382 (e/dm³ mol^-1 cm^-1, 15532), 365 (15700), 301(35887), 289 (30504), 278 (sh), 260 (sh), 254 (61008) ;_v_max (DCM mull)/cm^-1 3250- 2850 ⁺NH₃);_δ_H(250 MHz; CDCl₃) 8.75 (4H, m), 8.63 (2H, d, J7), 8.15 (2H, m),7.62 (8H, m), 7.39 (2H, d, J8, tosyl-H), 6.79 (2H, d, J8, tosyl-H), 4.97 (1H, t, J4), 2.47 (2H, m), 2.20 (2H, m), 1.96 (3H, s), 0.86 (2H, m), 0.45 (4H, m), 0.23 (2H, m),; m/z (FAB) 637 (M\ 0.6%), 466 (100, M- C₇H₇SO₃) (Found: MH⁺ 638.27288. C₄₂H₄₀NO₃S requires

638.27287).

Preparation of N-(3, 5 Dinitrobenzoyl)-α-phenylglycyl 6- (17-tetrabenzo[a,c,g,i]fluorenyl)hexyl amide 2b

This compound was obtained as above using amine salt 17b (136 mg, 0.21 mmol), R-N-(3,5- dinitrobenzoyDphenylglycine (83 mg, 0.24 mmol), EEDQ

(58mg, 0.24 mmol) and THF (5cm³). The product was isolated from the crude reaction mixture using dry flash

chromatography eluting initially with DCM then 0.2%

MeOH/DCM. This gave the amide 2b as an orange solid (135 mg, 80%), m.p. 141-143oC (Found: C, 75.38; H, 5.65; N, 7.01. C₅₀H₄₀N₄O₆ requires C, 75.74; H, 5.09; N, 7.07%); [α] _D at 25oC=-31.9o (c=6.4xl0^-3 in CHCl₃);_λ_max (CHCl₃) /nm 382

(e/dm³mol^-1cm^-1, 17056), 365 (17798), 302 (20764), 289

(35596), 278 (sh), 262 (sh), 254 (82315), 240 (70449);- _v_max(DCM)/cm^-1 3402,3293,3088 (NH), 1647 (C=O), 1540, 1343 (NO₂);_δ_H(250 MHz; CDCl₃) 8.96 (1H, t, J2), 8.76-8.70 (6H, m), 8.61 (3H, td, J7 and 1), 8.19 (2H, m), 7.62 (8H, m), 7.20 (2H, dd, J7 and 2), 7.08-6.94 (3H, m), 5.40 (1H,d,J6) 5.15 (1H,t,J6), 4.99 (1H,t,J4), 2.81-2.65 (2H,m,AB system), 2.54 (2H,m), 0.76 (2H, quin., J7 ) , 0.59 (2H,q,J7), 0.47 (2H,q,J7), 0.20 (2H, m) ;_δ_c (62.9 MHz; CDCl₃) 169.58

(4o,C=O), 161.20 (4o,C=O), 147.84 (2x4o), 144.06(4o),

144.02(4o), 137.13(4o), 136.56 (2x4o), 136.20(4o),

130.98(4o), 130.15(4o), 130.07(4o), 128.85(3o), 128.52(4o), 127.78(4o), 127.72(2x3o), 127.45 (2x3o), 127.28 (2x3o), 127.23(2x3o), 126.93 (2x3o), 126.76 (2x3o), 125.87(2x3o), 125.66(4o) , 125.03(2x3o) , 124.98(4o) , 124.34(2x3o) ,

124.29(4o) , 123.34(4x3o) , 120.62(3o) , 57.35(3o) , 46.84(3o) , 39.57(2o) , 33.20(2o) , 28.82(2o) , 28.58 (2o) , 25.73 (2o) , 21.85(2o) ; m/z (FAB) 793 (MH^*, 9%) , 466(4) , 365 (10) (Found: M^* _f 792.29478. C₅₀H₄₀N₄O₆ requires 792.29477) .

Example 3

Preparation of N-(3,5 Dinitrobenzoyl)-α-phenylglycyl 10- (17-tetrabenzo [a, c,g,i]fluorenyl) decylamide 2c

Preparation of Di-tert-butyl N-(10-bromodecyl)

aminodicarboxylate 15c

This compound was obtained by the above procedure using di-tert-butyl iminodicarboxylate (4.44 g, 20.5 mmol), sodium hydride (682 mg of an 80% dispersion in oil, 22.7 mmol), 1,10-dibromodecane (25.0 g, 83.3 mmol), THF (60 cm³) and DMF (60 cm³). After distillation the crude reaction mixture was dry flash chromatographed eluting initially with 2% ether/hexane. This gave 15c as a colourless oil (3.35 g, 37%);_v_max (neat)/cm^-1 2980, 2932, 2855 (CH), 1789, 1748, 1699 (C=O);_δ_H (250 MHz; CDCl₃) 3.41 (2H, t, J7), 3.26 (2H, t, J7), 1.71 (2H, quin, J7), 1.37 (18H, s, C(CH₃)₃)), 1.35-1.10(14H, m);_δ_c (62.9 MHz; CDCl₃) 152.34 (2x4°,

C=O),. 81.51 (2x4°, C(CH₃)₃), 46.11 (2°), 33.50(2°),

32.49(2°), 29.11 (2°), 29.00 (2°), 28.92(2°), 28.68 (2°), 28.40 (2°), 27.86 (6x1°, C(CH₃)₃)), 26.44 (2°); m/z (FAB) 435 (M⁺, 0.2%), 380 (10), 324 (67), 246 (65), 56 (100)

(Found: M⁺, 435.19844. C₂₀H₃₈ ⁷⁹BrNO₄ requires 435.19845. M⁺, 437.19649. C₂₀H₃₈ ⁸¹BrNO₄ requires 437.19648). Preparation of Di-tert-butyl N- (10- (17- tertrabenzo [a,c,g,i]fluoienyl)decyl) aminodicarboxylate 16c

This compound was obtained by the above procedure using Tbf 12b (2.8 g, 7.6 mmol) in 1, 4-dioxane (50 cm³), 40% aqueous ⁿBu₄N0H (5.0 cm³, 7.5 mol) in 1, 4-dioxane (14 cm³) and di-tert-butyl N-(10-bromodecyl) aminodicarboxylate 15c in 1, 4-dioxane (120 cm³). The reaction was complete after 2 hours at reflux. The crude reaction mixture was dry flash chromatographed eluting initially with 50%

DCM/hexane. This gave the product 16c a pale yellow solid (2.31g, 53%), m.p. 53-56°C (Found: N, 2.04 C₄₉H₅₅NO₄ requires N, 1.94%);_λ_max (DCM)/nm 382 (e/dm³ mol^-1 cm^-1, 18464), 365 (18904), 301 (42645), 289 (36050), 278 (sh), 260 (65066), 254 (71220), 240 (53196) ;_v_max (DCM mull) /cm^-1 2981, 2932, 2857 (CH), 1782, 1742, 1693 (C=O) ;_δ_H (250 MHz ; CDCl₃) 8.85- 8.80 (4H, m), 8.70 (2H, dd, J8 and 2), 8.21 (2H, dd, J8 and 2) 7.71-7.58 (8H, m), 4.92 (1H, t, J4), 3.48 (2H, t, J7) , 2.61-2.55 (2H, br.m), 1.49 (18H, s, C(CH₃)₃), 1.24-0.72

(14H, m) , 0.40-0.30 (2H, br.m) ;_δ_c (62.9 MHz ; CDCl₃) 152.59 (2x4°, C=O) , 144.28 (2x4°) , 136.68 (2x4°) , 131.18 (2x4°) , 130.29(2x4°) , 128.73 (2x4°) , 127.96(2x4°) , 127.39 (2x3°) , 126.68 (2x3°) , 125.77 (2x3°) , 125.53 (2x3°) , 124.92 (2x3°) , 124.10(2x3°) , 123.41 (4x3°) , 81.81 (2x4°, C(CH₃)₃) ) , 47.09 (3°) , 46.38 (2°) , 33.47(2°) , 29.37 (2°) , 29.15(2°) , 29.04 (2°) , 28.94 (2°) , 28.85 (2°) , 28.73 (2°) , 27.98 (6x1°, C(CH₃)₃)) , 26.57 (2°) , 22.12 (2°) ; m/z (FAB) 721 (M⁺, 50%) , 566 (21) , 522 (12) , 365 (79) , 56 (100) , (Found: M⁺,

721.41313. C₄₉H₅₅NO₄ requires 721.41309) .

Preparation of 10-(17-

Tetrabenzo [a,c,g,i]fluorenyl)decylamine p-toluenesulfonic acid salt 17c

This compound was obtained by the above procedure using di-tert-butyl N-(10-(17- tetrabenzo[a,c,g,i]fluorenyl)decyl) aminodicarboxylate 16c

(351 mg, 0.48 mmol), p-toluenesulfonic acid monohydrate (93 mg, 0.49 mmol) and DCM (25 cm³). It proved impossible to purify 17c using the above procedure and was used as isolated from the reaction mixture.

Preparation of N-(3, 5 Dinitrobenzoyl)-α-phenylglycly 10- (17-tetrabenzo [a,c,g,i]fluorenyl)decylamide 2c

This compound was obtained as above using free amine (164 mg, 0.31 mmol), R-N-(3, 5-dinitrobenzoyl)phenylglycine (117 mg, 0.34 mmol), EEDQ (83 mg, 0.34 mmol) and THF (6 cm³). The product was isolated from the crude reaction mixture using dry flash chromatography eluting initially with DCM then 0.2% MeOH/DCM. This gave the amide 2c as an orange solid (210 mg, 79%), m.p. 125-127.5oC (Found C: 76.26; H, 6.01; N, 6.59. C₄₅H₄₈N₄O₆ requires C, 76.40; H, 5.70; N, 6.60%); [α] _D at 23oC=-35.1o (c=6.8x10^-3 in

CCl ₃);_λ_max (CHCl₃)/nm 382(6/dm³mol^-1cm^-1, 17926), 365(18706), 302(42088), 290(36632), 260 (βh), 254 ( 82618 ) ;_v_max (DCM mull) /cm^-1 3398, 3306, 1639 (C=O), 1542;_δ_H (250 MHz; CDCl₃) 9.12(1H,d, J7), 8.90(1H,t, J2), 8.78-8.62 (8H,m),

8.19(2H,t, J6.5), 7.70-7.56(8H,m), 7.40-7.36 (2H,m), 7.27- 7.20 (3H,m), 5.79 (1H, t, J6 ), 5.67 (1H, d, J7), 4.93 (1H, t, J4), 3.20-2.95 (2H, m, AB system), 2.55-2.45 (2H, br.m), 1.28- 1.17 (2H, br.m), 0.95-0.6 (12H, m), 0.28 (2H,

br.s) ;_δ_c(62.9 MHz; CDCl₃) 169.76(4o) , 161.37(4o) ,

147.86(2X4o) , 144.23(4o) , 144.10(4o) , 137.21(4o) ,

136.51(4o) , 136.45(4o) , 136.26(4.0) , 130.93 (2x4o) ,

130.08(4o) , 130.03(4o) , 128.92 (2x3o) , 128.51 (4o and 3o) , 127.75(4o) , 127.72(2x4o) , 127.48(2x3o) , 127.21(2x3o) , 126.97(2x3o) , 126.61(2x3o) , 125.70(2x3o) , 125.46(2x3o) , 124.87(2x3o) , 124.32(3o) , 124.27(3o) , 123.27(4x3o) ,

120.60(3o) , 57.52(3o) , 46.91(3o) , 39.88(2o) , 33.37(2o) , 29.23(2o) , 28.81(3x20) , 28.59(2x2o) , 26.23 (2o) , 22.01(2o) ; m/z (FAB) 849 (MH⁺, 66%) , 523 (28) , 365 (100) (Found: M⁺, 848.35737. C₅₄H₄₈N₄O₆ requires 848.35737) .

(R,R)-O,O-diacetyltartaric acid mono (isopropylamide) was prepared as described in the literature (5.709g, 56%)mp 166-167°C(dec) (lit mp 176.5-177°C (dec))

The amine salt 17b (500mg, 0.78mmol) was dissolved in methanol (4ml) and DCM (20ml). This mixture was then washed with 0.4M NaOH aqueous. (5ml), water (10ml) and brine (5ml). It was dried with MgSO₄ and filtered.

Removal of solvent gave the free amine 15(X=NH₂) as a foam, which was used without further purification.

The free amine 15(X=NH₂) and (R,R)-O,O-diacetyltartaric acid mono(isopropylamide) (229mg, 0.83mmol) were dissolved in THF (25ml) under an atmosphere of nitrogen. EEDQ

(208mg, 0.84mmol) was added and the mixture stirred at room temperature for 24 hours. Solvent was removed at reduced pressure. the resulting yellow solid was dissolved in DCM (4ml) and hexane (25ml) was added. The yellow precipitate was filtered off and washed with hexane (2x). The above was repeated 2x. The yellow solid was dry flash

chromatographed using Kieselgel H, eluting initially with DCM and then 1% MeOH/DCM. The diacetate 18 was obtained as a yellow foam (464mg, 81%) mp 140-143°C.

Found (%) H 6.72:N 3.77 C₄₆H₄₆N₂O₆ requires H 6.42:N 3.88.

[α]_D and [α]₅₄₆ at 24°C (c= 10.2x10^-3 in CHCl₃) = -4.60° and - 4.43° respectively.

λmax(DCM) - 382 (e 16814), 365 (17308), 301 (38078), 288 (32144), 276 (s), 260 (58353), 254 (63793), 238 (s).

v/max (DCM mull, cm^-1) - 3288 (NH), 2978, 2938 (CH), 1755 (ester C=O), 1650 (amide C=O)

¹H (250 MHz, CDCl₃, δ) - 8.78 (4H, m) 8.66 (2H, dd, 8 and 1 Hz), 8.20 (2H, d, 6.5Hz), 7.71-7.52 (8H, m), 6.08 (1H, m, NH), 5.96 (1H, m, NH), 5.45 (1H, d, 3.5Hz), 5.40 (1H, d, 3.5Hz), 4.98 (1H, m), 5.40 (1H, m, CH(CH₃)₃), 2.84 (1H, sept, 7Hz), 2.73 (1H, sept, 7Hz), 2.55 (2H, m), 2.02 (3H, s, COCH₃), 1.88 (3H, s, COCH₃), 1.05 (2H, dd, 7Hz, AB system), 0.91 (2H, m), 0.70 (4H, m), 0.30 (2H, m) .

1³C (62.9MHz, CDCl₃, δ) - 169.01 (4°, C=O), 168.92 (4% C=O), 165.69 (4o, C=O), 155.00 (4º C=O), 144.07 (2x4º), 136.67 (2x4°), 131.16 (2x4°), 130.27 (2x4°), 128.63 (2x4º), 127.89 (2x4°) , 127.35 (2x3°) , 126.75 (2x3°) , 125.85 (2x3°) , 125.60 (2x3°) , 124.96 (2x3°) , 124.31 (2x3°) , 123.41 (4x3°) , 72.22 (3°) , 72.16 (3°) , 46.95 (3o) , 41.48 (3°) , 39.12 (2°) , 33.30 (2º) , 28.92 (2°) , 28.69 (2°) , 25.93 (2°) , 22.28 (1°) , 22.11 (1°) , 21.98 (2°) , 20.40 (1°) , 20.33 (1°) .

m/z (FAB) - 722 (M⁺, 52%) , 366 (25.9%), 365 (43.9%) , 364 (43.0%) , 363 (36.5%) , 44 (100%) .

HRMS - Found: M⁺, 722.33275, C₄₆H₄₆N₂O₆ requires 722.33559.

The diacetate 7 (495mg, 0.69mmol) was dissolved in methanol (30ml) with rapid stirring under an atmosphere of nitrogen. Potassium carbonate (493mg, 3.57mmol) was added followed by water (1ml) dropwise. The mixture was stirred at room temperature for 2 hours. Most of the solvent was removed at reduced pressure. DCM (50ml) was added. This was then washed with water (2x20ml) and brine. It was dried with MgSO₄ and filtered. The solvent was removed at reduced pressure to give a yellow solid. This was dry flash chromatographed, eluting initially with 2% MeOH/DCM. This gave the diol 4 as a yellow solid (402mg, 92%) mp 94-97°C. [α]D and [α]₅₄₆ at 24°C (c=10.8x10^-3 in CDCl₃) - -5.83° and - 5.98° respectively.

λmax (DCM:MeOH 1:1) - 382 (e 14781), 365 (15125), 302

(34375), 288 (29219), 277 (27500), 262 (53281), 254 (58438) 238 (s).

v/max (DCM mull, cm^-1) - 3396, 3302 (OH, NH), 2934, 1650 (amide C=O), 1537. ¹H (250 MHz, CDCl₃, δ) - 8.81-8.76 (4H, m), 8.67 (2H, dd, 8 and 1.5Hz), 8.22 (2H, dd, 7 and 2Hz), 7.71-7.57 (8H, m), 6.82 (1H, d, 8Hz, NH), 6.75 (1H, t, 6Hz, NH), 5.3 (1H, m, exchanges in D₂O.OH), 4.97 (1H, t, 4Hz), 4.17-4.07 (2H, m, 2xCHOH), 3.87 (1H, m, NCH(CH₃)₂), 2.79 (2H, q, 7Hz), 2.60- 2.52 (2H, m), 1.07 (3H, d, 6.5Hz), 0.94 (3H, d, 6.5Hz), 0.95-0.85 (2H,m), 0.71 (4H, br.m), 0.32 (2H, br.m).

1³C (62.9 MHz, CDCl₃, δ) - 172.32 (4°), 171.68 (4°), 143.89 (2x4°), 136.43 (2x4°), 130.97 (2x4°), 130.07 (2x4°), 128.40 (2x4°), 127.70 (2x4°), 127.17 (2x3°), 126.50 (2x3°), 125.63 (2x3°), 125.40 (2x3°), 124.76 (2x3°), 124.14 (2x3°), 123.24 (4x3°), 71.37 (2x3°, br.), 46.67 (3°), 41.02 (3°), 38.65 (2°), 33.02 (2°), 28.63 (2°), 28.41 (2°), 25.68 (2°), 22.19 (1°), 22.03 (1°), 21.64 (2°).

m/z (FAB) - 639 (MH⁺ 97.7%), 638 (M⁺ 32.0%), 466 (41.4%), 365 (92.2%), 364 (100%), 363 (99.2).

HRMS - Found M⁺ 638.31450 C₄₂H₄₂N₂O₄ requires 638.31446.

Example 6

Preparation of Dicarbamate 8

The diol 4 (59mg, 0.09mmol) was dissolved in pyridine (1ml) under nitrogen. Phenyl isocyanate (0.150ml) was added and the mixture stirred at room temperature for 5 hours. The pyridine was removed at reduced pressure (ImmHg). The resulting yellow glass was triturated with hexane, ether and DCM. The resulting yellow solid was adsorbed onto silica and dry flash chromatographed using Kieselgel H, eluting initially with DCM and then 1% MeOH/DCM. The dicarbamate 8 was obtained as a yellow solid (67mg, 8%) . Nmr shows this product is not pure (impurity appears to be diphenyl urea).

The dibenzoylate 9 was prepared as described above using the tosic acid salt 17b (296mg, 0.46mmol),

commercially available (FLUKA) (-)-O, O-benzoyl-L-tartaric acid mono-(dimethylamide) (190mg, 0.49mmol), EEDQ (132mg, 0.53mmol). The resulting solid was dry flash

chromatographed using Kieselgel H, eluting initially with DCM and then 1% MeOH/DCM. This gave the dibenzoylate 9 as a yellow foam (368mg, 95%) .mp 103-106°C.

Found (%) : H, 5.67 :N, 3.30, C₅₅H₄₈N₂O₆ require H, 5.81 :N, 3.36. [α]_D and [a]₅₄₆ at 24°C (c=8.85xl0^-3 in CHCl₃) - +7.34° and +9.72° respectively.

λmax (CCl ₃) - 382 (e 14689), 365 (15277), 301 (34079), 289 (30556), 266 (54644), 256 (60520).

v/max (DCM mull, cm^-1) - 3443, 3330 (NH), 2939 (CH), 1730 (ester C=O), 1673 (amide C=O).

1(250 MHz, CDCl₃, δ) - 8.77 (4H,m), 8.66 (2H, d, 8Hz), 8.16 (2H, m), 7.94 (4H, m), 7.65 (8H, m), 7.50 (1H, m), 7.40-7.23 (7H, m), 6.24 (1H, br. s, NH), 6.22 (1H, d, 6Hz), 5.88 (1H,d, 6Hz), 4.88 (1H, m), 3.14 (3H, s, NCH₃), 2.86 (2H, m), 2.77 (3H, s, NCH₃), 250 (2H, m), 0.90 (2H, m), 0.67 (4H, m), 0.26 (2H, m).

¹³C (62.9 MHz, CDC13),δ) - 165.56 (4°,C=O), 165.46 (4°,C=O), 165.11 (4°,C=O), 165.08 (4°,C=O), 143.8 (2x4º), 133.26 (3°). 133.05 (3°), 130.85 (2x4°), 129.94 (2x4°), 129.56 (2x3°), 129.44 (2x3o), 128.49 (4°), 128.39 (4°), 128.14 (2x3°), 127.96 (2x3°), 127.55 (2x4°), 127.03 (2x3°), 126.46 (2x3°), 125.58 (2x3°), 125.35 (2x3°), 124.70 (2x3°), 124.07 (2x3°), 123.19 (2x3°), 123.13 (2x3°), 72.37 (3°), 69.09 (3°), 46.54 (3°), 38.88 (2°), 36.70 (1°, NCH₃), 35.45 (1°, NCH₃), 32.93 (2°), 28.60 (2°), 28.52 (2°), 25.54 (2°), 21.56 (2°).

m/z (FAB) - 833 (MH⁺, 7.4%), 832 (M⁺, 3.4%), 365 (8.6%),

217 (6.1%), 126 (4.2%), 105 (100%), 91 (14.5%), 77 (12.1%). HRMS - Found: M⁺ . 832.35128. C₅₅H₄₈H₂O₆ requires 832.35124.

Example 8

Preparation of pentahvdroxy amide 5.

The free amine 15 (X=NH₂) was obtained from the tosic acid salt 17b (516mg, 0.81mmol) as above.

The free amine was dissolved in 1, 4-dioxane under

nitrogen. Methanol (19ml) and gluconic acid δ lactone (177mg, 0.99mmol) were added and the mixture refluxed for 18 hours. On cooling the solvent was removed. The

resulting solid was boiled with DCM (4ml) and solvent decanted. The above was repeated. The solid was boiled with water (4ml) and solvent decanted. The above was repeated. DCM was added and the solid filtered. The resulting yellow solid was adsorbed onto silica and dry flash chromatographed using Kieselgel H, eluting initially with 2% MeOH/DCM. The product 24 was obtained as a yellow solid (195mg, 37%)mp 117-120°C.

Found (%) : H.6.41: N.2.09. C₄₁H₄₁NO₆ requires H.6.42: N.2.18. [α]_D and [α]₅₄₆ at 24°C (c-6.1x10^-3 in CDCl₃.MeOH 1:1) +

16.39° and +20.49° respectively.

λmax (CHCl₃:MeOH 1:1) - 382 (e 21503), 365 (22418), 302 (47581), 288 (43921), 262 (75031), 254 (81436), 240 (s). v/max (DCM mull, cm^-1) - 3600-3100 (br., OH), 1644 (amide C=O).

¹H (250 MHz, CDCl₃/d₃-MeOD, δ) - 8.70 (4H, m), 8.56 (2H, d, 8Hz), 8.12 (2H, dd, 6 and 2Hz), 7.62-7.49 (8H, m), 4.88 (1H, s), 3.97 (1H, d, 3Hz), 3.86 (1H, t, 2Hz), 3.31 (2H, s), 2.81-2.60 (2H, m, AB system), 2.49 (2H, m), 0.85 (2H, q, 7Hz), 0.63 (4H, m), 0.20 (2H, m).

¹³C (62.9 MHz, CDCl₃/d₃-MeOD, δ) - 172.31 (4°, C=O), 143.57 (2x4°) , 135.98 (2x4°) , 130.63 (2x4°) , 129.72 (2x4°) , 128.03 (2x4°) , 127.30 (2x4°) , 126.76 (2x3°) , 126.15 (2x3°) , 125.29 (2x3°) , 124.94 (2x3°) , 124.34 (2x3°) , 123.83 (2x3°) , 122.90 (2x3°) , 122.83 (2x3°), 73.29 (3°), 72.36 (3°) , 70.90 (3°), 69.50 (3°) , 62.85 (2°) , 46.25 (3°) , 38.21 (2°), 32.75 (2°) , 28.44 (2°) , 28.14 (2°) , 25.50 (2°) , 21.44 (2°) .

m/z(FAB) - 644 (M⁺, 69.5%) , 365 (18%) , 217 (82.2%) , 109 (34%) , 91 (100%) .

HRMS - Found: M⁺, 643.29328. C₄₁H₄₁NO₆ requires 643.29339.

Example 9

Preparation of glycyl-L-proline derivative 3

The ammonium salt of Tbf (13) was reacted with 6- bromohexan-1-ol in refluxing dioxane to give the alcohol (15, X=OH) in 35% yield. The alcohol 15 was then reacted with triphosgene in DCM and N,N-dimethylaniline giving the chloroformate in 95% yield, which reacted cleanly with commercially available Glycol-L-proline in Na₂CO₃, H₂O and dioxane giving Tbfhexanoxycarbonylglycyl-L-proline 3 in 80° yield.

Example 10

Preparation of ion pair 6

Amine salt 17b was adsorbed onto PGC and treated with NaOH(aq) to give the free amine and then R N-(3,5- dinitrobenzyl)phenyl glycine to give ion pair 6.

Chambers containing PGC coated with a compound of the present invention generally show high stability. Even if pumped through with solvent for many days, separation of enantiomers may still be achieved.

Example 11

Preparation of tert-butyl 6-bromohexanoate 21

2-Methyl propan-2-ol (16.04 g, 0.21 mol) and pyridine (12 cm³) were dissolved in DCM (75 cm³) under nitrogen and cooled to 0°C. 6-Bromohexanoyl chloride (14 cm³, 91.5 mmol) was added and the mixture stirred at 0°C for 1 hour and room temperature for 15 minutes. 2N HCl (75 cm³) was added and the separated aqueous layer extracted with ether (2x50 cm³). The combined organic layers were washed with water, 2N NaOH (aq), water and brine. It was dried with MgSO₄ and filtered. Removal of solvent at reduced pressure gave an orange oil. This was distilled and gave the product as a colourless liquid 14.91g, 65%, bp 84-94 °C at 0.6 mmHg. v_max (neat)/cm^-1 1729 (C=O); δ_H (250 MHz; CDCl₃) 3.36 (2H, t, J7, CH₂Br), 2.16 (2H, t, J7, CH₂CO), 1.82 (2H, q, J7 ), 1.56 (2H, m), 1.145-1.35 (11H, m, includes at 1.40 9H, s, C(CH₃)₃); δ_c (62.9 MHz; CDCl₃) 172.63 (4°, C=O), 79.91 (4°, C(CH₃)₃), 35.10 (2°), 33.35 (2°), 32.25 (2°), 27.91

(3x1°), 27.34 (2°), 24.02 (2°); m/z (FAB) 252 (M⁺ (⁸¹Br) , 4.1%), 250 (M⁺ (⁷⁹Br), 5.7), 234 (13.5), 218 (11.3), 197 (16.1), 195 (11.3), 133 (15.7), 130 (10.6); (Found: MH⁺ 251.06433. C₁₀H₂₀ ⁷⁹BrO₂ requires 251.06467. Found:

MH⁺ 253.06330. C₁₀H₂₀ ⁸¹BrO₂ requires 253.06275).

Preparation of tert-butyl 6-(17- tetrabenzo[a,c,g,i]fluorenyl)hexanoate 22

The Ammonium salt, 13, was prepared as described before using Tbf (1.23 g, 3.37 mmol), 1.5M ⁿBu₄NOH (2.25 cm³) and degassed 1,4 dioxane (57 cm³) and it was then mixed with 21 (0.850 g, 3.38 mmol) in degassed 1,4 dioxane (25 cm³). The mixture was refluxed for 30 minutes before all the solid dissolved and for a further 15 minutes. The mixture was cooled and solvent removed at reduced pressure. Ether (25 cm³) was added and the mixture triturated. The solid was filtered off and discarded. The solvent of the filtrate was removed to give a red oil. This was triturated with hexane before it was purified using dry flash

chromatography, eluting initially with 50% DCM/hexane.

This gave the product 22 as a yellow solid 1.150 g, 63%, mp 63-66°C. (Found: C, 87.40; H, 7.17. C₃₉H₃₆O₂ requires C, 87.28; H, 6.76%). λ_max (CHCl₃)/nm 380 (ε/dm³ mol^-1 cm^-1, 12201), 364 (12342), 302 (33147), 290 (35333), 260 (56703), 256 (57831); v_max (DCM mull)/cm^-1 1725 (C=O); δ_H (250 MHz; CDCl₃) 8.82 (4H, m), 8.68 (2H, dd, J6.5 and 1.5), 8.27 (2H, m), 7.74-7.58 (8H, m), 5.09 (1H, t, J4.5), 2.64 (2H, m), 1.73 (2H, t, J7), 1.54 (3H, s), 1.25 (6H, s), 1.03 (2H, m), 0.97 (2H, m), 0.39 (2H, m); δ_c (62.9 MHz; CDCl₃) 172.75 (4°), 143.99 (2x4°), 136.58 (2x4°), 131.09 (2x4°), 130.20 (2x4°), 128.55 (2x4°), 127.82 (2X4°), 127.28 (2x3°), 126.60 (2x3°), 125.69 (2x3°), 125.45 (2x3°), 124.81 (2x3°), 124.22 (2x3°), 123.33 (2x3°), 123.31 (2x3°), 79.43 (4°), 46.84 (3°), 34.97 (2°), 33.26 (2°), 28.75 (2°), 27.77 (3x1°), 24.19 (2°), 21.80 (2°); m/z (FAB) 536 (M⁺, 41.8%), 364 (36.6), 363

(47.2), 57 (85), 41 (100); (Found: M⁺ 536.27154. C₃₉H₃₆O₂ requires 536.27153).

Preparation of 6-(17-tetrabenzo[a,c,g,i]fluorenyl)hexanoic acid 23

The ester 22 (0.620 g, 1.16 mmol) was dissolved in DCM (2cm³) under nitrogen. TFA (2cm³) was added and the

mixture stirred at room temperature for 1 hour. Solvent was removed at reduced pressure to give a red solid. This was dissolved in DCM (50 cm³) and washed with water (3x) and brine. It was dried with MgSO₄ and filtered. Solvent was removed and the mixture dry flash chromatographed eluting initially with 90% DCM/hexane. This gave the product 23 as a yellow foam 519g, 93% mp 110-113°C. (A larger scale reaction gave material as a yellow solid mp 183-186°C, with identical proton nmr). (Found: C, 82.90; H, 5.97. C₃₅H₂₈O₂ requires C, 87.47; H, 5.87%); λ_max (CHCl ₃) /nm 382 (ε/dm³ mol^-1 cm^-1, 13786), 366 (14061), 302 (32035), 290 (29730), 256 (57029); v_max (DCM mull) /cm^-1 1705 (C=O); δ_H (250 MHz; CDCl₃) 8.79 (4H, m), 8.67 (2H, dd, JB and 1.5), 8.26 (2H, m), 7.73-7.58 (8H, m), 5.08 (1H, t, J4.5), 2.66- 2.58 (2H, m), 1.81 (2H, t, J7), 1.04 (2H, quin, J7), 0.81 (2H, quin, J7 ), 0.39-0.32 (2H, m); δ_c (62.9 MHz; CDCl₃) 179.78 (4°), 143.89 (2x4°), 136.53 (2x4°), 131.04 (2x4°),

130.15 (2x4°), 128.47 (2x4°), 127.77 (2x4°), 127.24 (2x3°), 126.57 (2x3°), 125.67 (2x3°), 125.45 (2x3°), 124.81 (2x3°),

124.16 (2x3°), 123.32 (2x3°), 123.30 (2x3°), 46.71 (3°), 33.34 (2°), 33.08 (2°), 28.58 (2°), 23.60 (2°), 21.68 (2°); m/z (FAB) 480 M⁺, 100%), 364 (84.7), 363 (43.5), 154 (75), 41; (Found: M⁺ 480.20896. C₃₅H₂₈O₂ requires 480.20893).

Preparation of Vancomycin Derivative 11

The acid 23 (0.577g, 1.20 mmol) and N-hydroxysuccinimide (0.169g, 1.47 mmol) were dissolved in THF (7 cm³) under nitrogen. N,N'-DiiDopropylcarbodiimide (0.225 cm³, 1.46 mmol) was added and the mixture stirred at room temperature for 4 hours. Ether (15 cm³) was added and the white precipitate filtered off and washed with ether (2x). The combined organic layers were washed with water (2x), sat^d NaHCO₃ (aq), water and brine. It was dried with MgSO₄ and filtered. Removal of solvent gave a yellow foam. This was dry flash chromatographed eluting with 80% DCM/hexane.

This gave as a yellow foam 0.436 g, 63%. v_max (DCM mull)/cm^-1 1812, 1785, 1740; δ_H (250 MHz; CDCl₃) 8.80 (4H, m), 8.68 (2H, d, J7 ), 8.27 (2H, dd, J7 and 1.5), 7.74-7.59 (8H, m), 5.10 (1H, t, J4.5), 2.72 (4H, s), 2.69-2.62 (2H, m), 1.95- 1.74 (2H, m, AB system), 1.28-1.15 (2H, m), 0.96-0.87 (2H, m), 0.43-0.36 (2H, m). Both the proton and carbon 13 nmr show trace impurities.

Vancomycin (283.8 mg, 0.182 mmol) was suspended in DMF (5 cm³) under nitrogen. 0.248M NaOH (aq) 1.2 cm³ was added and the mixture stirred at room temperature for 4 minutes. To the clear solution was added NaHCO₃ (s) (30 mg, 0.36 mmol) and the mixture stirred for 2 minutes. The active ester (80.1 mg, 0.139 mmol) was added and the mixture stirred for 24 hours. TFA (0.1 cm³) in water (10 cm³) was added and the mixture extracted with ether (4x50 cm³).

Care was taken during extraction to prevent the product precipitating out. The mixture was centrifuged to remove the final traces of ether and the aqueous layer was decanted off the white solid. The white solid was washed with water and dried giving 210 mg. v_max (DCM mull) /cm^-1 1670, 1657; λ_max 379, 362, 299, 287, 251, 236; m/z 1911.62 (M⁺) . C₁₀₁H₁₀₁Cl₂N₉O₂₅ requires 1911.86.

Example 12

Preparation of ((Boc)₂Lys)-₂-LysOMe

N-α, ε-di-tert-butoxycarbonyl-L-Lysine (3g, 8.66 mmol) was dissolved in THF (50 cm³) under an atmosphere of nitrogen. Et₃N (1.2 cm³, 8.61 mmol) was added and the mixture cooled to -5°C. Ethyl chloroformate (0.820 cm³, 8.61 mmol) was added dropwise at -5°C with stirring. The mixture was stirred at -5°C for 20 minutes.

L-Lysine methyl ester dihydrochloride (0.837g, 3.59 mmol) was dissolved in MeOH (20 cm³) and a methanolic solution of NaOH (0.95M, 7.5 cm³) was added. After

standing for 5 minutes the solvent was removed at reduced pressure. DCM was added and the mixture filtered. Solvent was removed to give the free diamine which was dissolved in THF (5 cm³ and 2x1 cm³) and added dropwise to the mixed anhydride prepared above at -5°C. The mixture was stirred at -5°C for 2 hours. Ether (50 cm³) was added and the mixture washed with 2M HCl, water, NaHCO₃ (aq), water and brine. It was dried with MgSO₄ and filtered. Removal of solvent at reduced pressure gave an oil. This was dry flash chromatographed eluting initially with 0.5% MeOH/DCM. This gave the product as a white solid 2.58g, 88%, mp 68- 70°C, [α]_D at 23.5°C -38.6° (c-0.018 in CC1 ₃). (Found: C, 56.46; H, 8.83; N, 10.25. C₃₉H₇₂N₆O₁₂ requires C, 57.34; H, 8.88; N, 10.29%). v_max (DCM mull)/cm^-1 3330, 3300, 1745, 1700, 1680, 1660; δ_H (250 MHz ; CDCl₃) 7.64 (1H, br.d,

J7.5), 7.28 (1H, br.s), 5.96 (1H, br.d, J7.5), 5.72 (1H, br.d J7.5), 4.92 (1H, br.s), 4.79 (1H, br.s), 4.42-4.34 (2H, m), 4.20 (1H, m), 3.66 (3H, s, OMe), 3.49-3.46 (1H, br.m), 3.1-2.85 (5H, m), 1.9-1.6 (6H, m), 1.55-1.1 (48H, m); δ_c (62.9 MHz; CDCl₃) 173.33 (4°), 173.28 (4°), 172.16 (4°), 156.14 (4°), 156.05 (4°), 155.74 (2x4°), 79.41 (4°), 79.34 (4°), 78.43 (4°), 78.32 (4°), 53.68 (3°), 53.19 (3°), 52.25 (3°), 51.83 (1°, OMe), 39.85 (2x2°), 38.26 (2°), 32.84 (2°), 32.50 (2°), 30.89 (2°), 28.96 (2°), 28.87 (2°), 28.53 (2°), 28.15 (12x1°, C(CH₃)₃), 22.93 (2°), 22.53 (2°),

22.18(2°); m/z (FAB): 817 (MH⁺, 0.1%), 417 (6.3, M-4xBoc), 84 (59), 57 (100, C₄H₉); Found: M⁺-H, 815.51298. C₃₉H₇₁N₆0₁₂ requires 815.51300).

Preparation of ((Boc)₂Lvs) -₂-Lys OH 24

((Boc)₂Lys)₂-Lys-OMe (788 mg, 0.96 mmol) was dissolved in dioxane (1.5 cm³) under nitrogen. 1M NaOH (1.1 cm³) was added and the mixture stirred at room temperature for 2 hours. The mixture was neutralised with 2M citric acid (0.55 cm³). The mixture was cooled to 0°C and 2M citric acid (2 cm³) was added. Ethyl acetate (40 cm³) was added and the separated organic layer washed with water (2x5 cm³) and brine. It was dried with Na₂SO₄ and filtered. Removal of solvent at reduced pressure gave a foam. This was dissolved in DCM and hexane was added. The solvent was decanted off the resulting oil and washed with hexane. The oil was dried at 0.3 mmHg. This gave the product 24 as an oily foam yield >100%! v_{ma x} (DCM mull) /cm^-1 3310, 1710, 1690, 1670, 1650, 1640. The nmr is hard to interpret since the peaks are broad and overlapping. The ¹H nmr signal

assigned to OMe group in starting material is no longer present . m/z (FAB) : 803 (MH⁺, 0 . 4%) , 403 ( 32 . 1 ) , 57 ( 79 . 5 ) . ( Found : MH⁺ 803 . 51478 . C₃₈H₇₁N₆O₁₂ requires 803 . 51300 ) . Preparation of 25

((Boc)₂Lys)₂-Lys-OH 24 (302 mg, 0.377 mmol) and Et₃N (0.060 cm³, 0.430 mmol) were dissolved in THF (12 cm³) under an atmosphere of nitrogen and cooled to -5°C. Ethyl

chloroformate (0.040 cm³, 0.42 mmol) was added and the mixture stirred at -5°C for 15 minutes. The free amine of 17b (196 mg, 0.427 mmol) was dissolved in THF (5 cm³ and 1 cm³) and added to the above mixture at -5°C. The mixture was stirred at -5°C-0°C for 45 minutes. Ether (30 cm³) was added and the mixture washed with 2M HCl, water and brine, it was dried with MgSO₄ and filtered. Removal of solvent at reduced pressure gave a yellow oil. This was dry flash chromatographed eluting initially with DCM. This gave the product 25 as a yellow solid 323 mg, 68%, mp 104-107°C,

[α]_D at 22°C (c = 10.4x10^-3 in CCl ₃) -39.13°. Found: C, 69.50; H, 8.23; N, 7.78. C₇₃H₉₉N₇O₁₁ requires C, 70.11; H, 7.98; N, 7.84%). λ_max (CHCl₃)/nm 380 (ε/dm³ mol^-1 cm^-1,

17864), 366 (18314), 302 (40457), 288 (35728), 254 (72583); v_max (DCM mull)/cm^-1 3340, 3280, 1690, 1640; δ_H (250 MHz;

CDCl₃) 8.80 (4H, m), 8.67 (2H, dd, J8 and 1), 8.26 (2H, dd,

J7 and 2), 7.8-7.6 (8H, m), 7.02 (1H, br.d, J6.5), 6.75

(1H, br.s), 6.12 (1H, br.s), 5.82 (1H, br.s), 5.38 (1H, br.m), 5.07 (1H, t, J4.5, 4.72 (2H, br.s), 4.2-3.9 (4H, br.m), 3.5-3.3 (1H, br.s) 3.1-2.95 (5H, br.m), 2.95-2.7 (2H, m), 2.60 (2H, m), 1.8-1.1 (50H, br.m), 1.00 (2H, br.m), 0.74 (4H, br.s), 0.32 (2H, br.m); (Found: M⁺

1249.74330. C₇₃H₉₉ N₇O₁₁ requires 1249.74026).

The following section illustrates the stability of compounds of the present invention on PGC and the different elution times of enantiomers: Test Examples

The structures of the compounds separated were: β

Chromatography

2-5 μl Samples of approximately 0.001% (w/v)

solutions of analytes were injected on to 100 x 4.6 mm columns packed with 7 μm porous graphite (Hypercarb^R) bearing, where desired various CSP's. Capacity factors, k', were determined using acetone as unretained marker. Chiral selectivities, a, were calculated as α_= k'₂ /k'₁ , where K'₁ and k'₂ are the k'-values of the first- and second-eluting enantiomers.

The resolutions achieved by the CSP's prepared from the amides 2 (denoted CSP 2's) when adsorbed on to porous graphite, are recorded for the aryl alcohols 26 in Table 1, and for the Fmoc amino acids 27 in Table 2.

Table 1 shows that CSP 2 (n' = 4) provides the highest selectivities for the separation of the aryl alcohols, and that isopropyl alcohol/hexane eluents give higher selectivities than ethyl acetate/hexane eluents.

Table 2 confirms that, with CSP 2 (n' = 4), the best resolution of the Fmoc amino acids is also obtained with the isopropyl alcohol containing eluents.

The stability of the CSP 2 phases to the eluent was tested using CSP 2 (n' = 10) by pumping isopropyl

alcohol/hexane (10/90 v/v) through the column continuously at 1 ml min^-1 for 20 days, corresponding to 20,000 column volumes. Over some 90 injections of 26c, selectivity, retention and peak shape were unchanged. The stability of the phase to protic solvents was tested using CSP 2 (n' = 6) by pumping isopropyl alcohol through the column as 1 ml min^-1 for 20 min, followed by water (20 min), isopropyl alcohol (30 min) and hexane. After re-equilibration with isopropyl alcohol/hexane (10/90 v/v), this column was able to separate, as before, 26b, 26i, and 27a showing a = 1.83, 1.29, 1.16 respectively (see Tables 1 and 2 for comparison with initial values).

It was noted that there was less broadening of the peaks the further the chiral selector was away from the tbf-group.

acetate/hexane (10/90 v/v): D ethyl acetate/hexane (5/95 v/v).

Table 2

Chiral Resolution of Racemic Fmoc Amino Acids 27 by CSP 2 (n' = 6)

a The L enantiomer generally eluted first, with the exceptions being asterisked. ^b Solvents used: A, isopropyl alcohol/hexane (10/90 v/v); B, isopropyl alcohol/hexane (5/95 v/v); C ethyl acetate/hexane (10/90 v/v); D ethyl acetate/hexane (5/95 v/v).

The diol-based CSP 4 adsorbed on to PGC resolved bi-β- naphthol using dichloromethane/hexane (5/95 v/v) as eluent with a = 1.06.

CSP 9 prepared from dibenzoyl tartrate, separated 9- anthrylphenyl methanol 26 using ethylacetate/hexane (5/95 v/v) or diethylether/hexane (10/90 v/v) as eluents with a = 1.09, k'₁ = 4.63 and a = 1.11, k'₁ = 4.20 respectively.

Table 3 shows the resolution of enantiomers of the aryl alcohols 26 by CSP 6

Table 3

Solvent: A = 10% isopropyl alcohol/hexane; B = 10% ethyl acetate/hexane; C = 10% ether/hexane.

To test the purity of the prepared sample of 11 (Vancomycin grafted to tbf), a small amount was subjected to analytical HPLC using a C-18 ODS silica reversed phase column under the gradient elution with the programme set out in Table 4, a flow rate of 1 ml min^-1 and detection at 364 nm.

Major compounds eluted after 18.4 and 27.9 min.

amounting to 70% and 12% of the injected material

respectively. All other compounds were present at <5%. Under the same conditions the active ester 8 eluted after 23.4 min.

Resolution of typical enantiomeric compounds using CSP 11 are listed in Table 5.

Preparation of multiple-linker CSP's

The multi-linker CSP 10 was prepared starting with the tetra-boc compound 25, of which 178mg, 0.143 mmol was initially dissolved in DCM (1 ml) under nitrogen. TFA (1 ml) was added with stirring and the mixture further stirred at room temperature for 45 min. Solvent was removed under reduced pressure. DCM was added twice and removed. The resulting red solid was dissolved in MeOH/DCM and washed with 2M NaOH (aq) (10 ml) to give the free amine, which was dried with Na₂SO₄ and filtered. Solvent was removed to give a yellow oil. This was dissolved in DCM/MeOH (50/50 v/v) (12 ml) and equal volumes added to R' N-3,5- dinitrobenzoyl-α-phenylglycine (R-DNBPG) (128 mg, 0.37 mmol) and S-DNBPG (132 mg, 0.38 mmol) both dissolved in THF (5 ml). The mixtures were stirred at room temperature for 1 hr. before solvent was removed under reduced pressure giving orange solids. These were dissolved in MeOH. PGC (1.147 g and 1.163 g) was added to the R and S solutions respectively. The mixtures were left to stand with occasional swirling. They were then centrifuged and excess solvent decanted. They were further washed three times with MeOH, centrifuged and the solvent decanted.

The CSP-loaded PGC was then packed into columns by standard methods known to those experienced in the art.

Table 6 compares the retention and resolution of racemic aryl alcohols 26 using the multiple-linker CSP 10, with those using the mono-linker CSP 6. It is seen that there is no consistent difference in retention between the two CSP's, and that, where the resolution is compared, the two CSP's give similar results.

aColumn: 6 ionic compound 6 adsorbed onto PGC; ionic compound 10 adsorbed onto PGC. bSolvent: A, ethyl acetate/hexane (10/90 v/v); B, 10% isopropyl alcohol/hexane (10/90 v/v).

Stripping and Loading Experiments

Two methods were used to load PGC with CSP's to provide the final chiral selective packing material, external loading, and, dynamic loading.

1) External Loading

The CSP was dissolved in the minimum amount of a poor to moderate solvent, and loose PGC added. The relative quantities of the CSP and PGC were chosen to give

approximately monolayer coverage of the internal graphitic surface of the PGC. The mixture was stirred for 1 hr, allowed to settle and the solvent decanted. The material was washed several times with an appropriate poor solvent for the CSP, which was likewise decanted. The PGC bearing the CSP was then packed into an HPLC column by standard methods. Various initial solvents for the CSP were examined: Acetonitrile was a suitable solvent for the method, although the low solubility of tbf-derivatives necessitated several hours stirring to dissolve the CSP. By using dicloromethane/acetonitrile/methanol mixture

(2/10/35 v/v) this disadvantage was overcome. The CSP was dissolved initially in the minimum quantity of

dichloromethane, and the appropriate quantities of

acetonitrile and methanol added subsequently. Both methods gave good chromatographic performance when the PGC loaded with the CSP was packed into the HPLC column, but the first method was the more reproducible.

2) Dynamic Loading

Slightly more than enough CSP to give monolayer coverage of the PGC was dissolved in acetonitrile at a concentration of about 100 mg/100ml. This solution was pumped through a column prepacked with PGC (a standard column packed with Hypercarb^R by Shandon HPLC) until the CSP could be detected emerging from the column. At this point the column was considered to be equilibrated with the CSP solution and to have adsorbed sufficient CSP to provide approximately monolayer coverage of the PGC surface. For this method it is important that the CSP is only sparingly soluble in the solvent so that once the CSP comes into contact with the graphitic surface it is strongly adsorbed.

In practice when the CSP was loaded as above using pure acetonitrile, it was found impracticable to wash out excess CSP using acetonitrile as this took more than 24 hr. Two methods were investigated to avoid this problem: (a) the column was loaded until it contained 95% of the

saturation level of CSP, and (b) following loading, the excess CSP was removed with a strong solvent pumped for a short time. The latter method was the more successful, as it was difficult, when using method (a), to know exactly when 95% loading had been achieved.

Stripping Experiments

Columns containing PGC loaded with the Pirkle material CSP 2, were treated with a range of solvents at different temperatures to determine the optimum conditions for removal of the CSP, and at the same time which solvents were acceptable for liquid chromatography (i.e. those which did not strip the CSP). The solvents were pumped through the PGC columns loaded with CSP 2 at a flow rate of 1 m/ min^-1 for 4 hr. The solvents are rated on a

semiquantitative scale from zero (no detectable stripping at room temperature) to 5 (over 60% stripping at room temperature). The results are set out in Table 7

The following conclusions may be drawn:

Solvents with stripping power 0 can be regarded as safe to use as eluents in HPLC when using graphites loaded with tbf-based CSP's. They gave no stripping at room temperature.

Solvents with stripping power 1 leach the CSP slowly, and can be used in eluents if mixed at low

concentration with class zero solvents.

Solvents with stripping power 2 or 3 cannot be used in eluents and are not useful for removing CSP's. They should be avoided.

Solvents with stripping power 4 readily remove most of the adsorbed CSP but are not suitable for total removal. They are unsuitable as chromatographic eluents or eluent components .

Solvents with stripping power 5 are useful for removing CSP's in order to regenerate PGC which can then be recoated with the same or another CSP.

Further Stripping Experiments - Effect of Temperature

Toluene and xylene have been identified as the strongest solvents for tbf-based CSP's. While the CSP can be completely stripped from PGC by unpacking the column, and refluxing the material with toluene, the CSP recovered from this procedure no longer showed enantiomeric

selectivity. Experiments were accordingly carried out heating with toluene at 20°C, 40°C and 70°C. The amount of CSP removed was determined by weighing. The stripped PGC was subsequently tested using a 6-peak test mixture (developed for Hypercarb) which is particularly sensitive to surface uniformity. The results are detailed in Table 8, of which the last column gives the quality of the chromatography using the 6 -peak test mixture.

Flow rate 0.2ml/min.

Table 8 shows that toluene and xylene behave similarly and completely strip CSP 2 at 70°C. The stripped PGC then behaves as does original PGC which has not been treated with the CSP. This is confirmed by the

chromatograms shown in Figures 2 and 3.

Unfortunately CSP 2 was found to degrade above 40°C in toluene. To avoid degradation it is therefore necessary to carry out the stripping in two stages if the CSP is to be reused. The recommended procedure is to remove the bulk of the CSP (about 90%) by heating with toluene at 40°C for 3-4 hr. and to complete the stripping at 70°C for 2 hr. The material recovered at 40°C can then be purified and reused. Overall toluene is superior to xylene since it can more readily be removed from the recovered PGC and CSP.

Phase Coverage

The number of moles of Pirkle groups per unit mass of support was determined for CSP 2 on PGC and for a silica- base Pirkle phase, as follows:

CSP 2 on PGC

Mass of CSP 2 per gm of PGC 47 mg

Mass contained within 100x4.6 mm column 35 mg

Quantity of Pirkle group per column 4.5 x 10^-5 mol Silica based Pirkle phase

% carbon in HS APS-2 silica 2.74%

% carbon in Pirkle silica column 6.70%

Quantity of Pirkle groups per gram of packing

3.0 x 10^-4 mol Quantity of Pirkle groups per standard 250 x 4.6 mm column

7.4 x 10^-4 mol Quantity of Pirkle groups per 100 x 4.6 mm column

3.0 x 10^-4 mol The PGC column contains about l/l6th the molar

quantity of Pirkle groups if standard size columns are considered, but on a per unit-volume-basis the PGC packing contains about 1/7th the molar quantity of Pirkle groups.

Claims

A compound of the formula:

Ar-L-(*)_m in which Ar represents a substantially planar, fused ring system which contains at least 4 aromatic rings and which is of formula:

wherein:

n is 0 or 1;

0 means that the surrounding ring is aromatic when n is 1 and non-aromatic when n is 0 ;

p is 0 or 1; and p is 0 when n is 0;

R₁ and R₂, on the one hand, and R₃ and R₄, on the other hand, are chosen so that each forms a fused aromatic ring system together with the carbon atoms to which they are attached;

R' is an alkyl group or hydrogen; and

R₅ is a group which, together with the atoms to which it is attached, forms one or more supplementary rings;

L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom;

* is a radical with at least one chiral centre; and m is 1, 2 or 3;

with the proviso that when * is a peptide or naturally occuring amino acid radical then the two chain atoms nearest the group Ar are carbon atoms.

2. A compound according to claim 1 in which L has from 2 to 10 chain atoms.

3. A compound according to claim 2 in which L has 6 chain atoms.

4. A compound according to any one of the preceding claims in which all the chain atoms are carbon atoms.

5. A compound according to any one of the preceding claims in which * is derived from a naturally occurring product.

6. A compound according to claim 6 in which * is derived from an antibiotic.

7. A compound according to any one of the preceding claims in which * is derived from phenyl glycine, glycyl-L- proline, tartaric acid, glucose, cellulose or gluconic acid.

8. A compound according to any one of claims 1 to 4 in which * is a Pirkle-type group.

9. A compound according to claim 8 in which the Pirkle-type group comprises a polar substituent, an

electron-poor system, an aromatic system which is not poor in electrons and a chiral carbon centre bearing a hydrogen atom and/or an alkyl group.

10. A compound according to claim 9 in which the polar substituent is an amide.

11. A compound according to any one of claims 1 to 4 in which * is derived from a cyclic peptide.

12. A compound according to any one of the preceding claims in which Ar is

13. A compound according to claim 1 specifically mentioned herein.

14. A process for the preparation of a compound according to claim 1 substantially as described in any one of Examples 1 to 14.

15. A compound according to claim 1 where prepared by the process of claim 14.

16. A graphite material on which is adsorbed a compound according to any one of claims 1 to 13 and 15.

17. A graphite material according to claim 16 which is graphite or activated charcoal.

18. A graphite material according to claim 15 or 16 substantially as hereinbefore described.

19. A process for the preparation of a graphite material according to claim 16, 17 or 18 which comprises treating the graphite material with a compound of formula Ar-L-(*)_m in which Ar, * and m are as defined in claim 1 and L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom.

20. A process according to claim 19 substantially as hereinbefore described.

21. A graphite material according to claim 16, 17 or 18 when prepared by the process of claim 19 or 20.

22. A chamber filled with a graphite material as claimed in any one of claims 16 to 18 or 21.

23. A chamber according to claim 22 in which the graphite material is graphite or activated charcoal.

24. A chamber according to claim 22 or 23

substantially as hereinbefore described.

25. A process for the preparation of a chamber according to claim 22, 23 or 24 which comprises either packing a graphite material according to claims 16, 17, 18 or 21 into a chamber or packing a graphite material into a chamber then passing a solution of a compound of formula

Ar-L-(*) in which Ar, * and m are as defined in claim 7 and L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom through the chamber.

26. A process according to claim 25 substantially as hereinbefore described.

27. A chamber according to claim 22, 23 or 24 when prepared by the process of claim 25 or 26.

28. A kit for separating enantiomers which comprises a compound as claimed in any one of claims 1 to 13 or 15, a chamber and a graphite material.

29. A kit according to claim 28 in which the graphite material is graphite or activated charcoal.

30. A kit according to claim 28 or 29 which also comprises a fraction-collecting assembly fitted with an ultraviolet (UV) or visible spectroscopic detector.

31. A process for the separation of enantiomers, which process comprises:

contacting a mixture of enantiomers with a compound of formula Ar-L-(*) in which Ar, * and m are as defined in claim 1 and L is a stable chain having at least two atoms in which the atoms are independently selected from carbon, oxygen, sulphur, nitrogen and phosphorus, where appropriate the chain atoms being optionally substituted by one or more C_1-6 alkyl groups which may be the same or different, one or more chain atoms forming part of one or more aromatic or heteroaromatic groups and where a carbon is adjacent to a nitrogen atom the carbon atom being optionally substituted by an oxygen atom simultaneously or successively, with a graphite material.

32. A process according to claim 31 in which the compound is adsorbed on the graphite material after being contacted with the mixture of enantiomers.

33. A process according to claim 31 in which the compound is adsorbed on the graphite material before being contacted with the mixture of enantiomers.

34. A process according to claims 31, 32 or 33 in which an enantiomer is retained by the compound.

35. A process according to claim 31 or 33 in which the enantiomers elute out of the chamber at different ratee.

36. A process according to any one of claims 31 to 35 in which the graphite material is graphite or activated charcoal.

37. A process according to any one of claims 31 to 36 substantially as hereinbefore described.

38. Use of a compound as defined in any one of claims 1 to 13 or 15 to separate enantiomers.