Synthesis and structure of a bis-glycosylated hexa- -peptide

The bis-glycosylated hexapeptide  -Ala-  -Ala-(  - D -Glc)- L -Asp-  -Ala-  -Ala-(  - D -Gal)- L -Asp ( 14 ) is prepared by fragment condensation of Fmoc- -Ala-  -Ala-(  - D -GlcAc 4 )- L -Asp-OH ( 11 ) and H 2 N- -Ala-  -Ala-(  - D -GalAc 4 )- L -Asp-O t Bu ( 12 ) under optimized conditions with the HBTU/HOBt reagent followed by deprotection of the intermediate fully protected hexapeptide. Hexapeptide 14 is shown to adopt a random coil structure in solution.


Introduction
Specific interactions of proteins with complex carbohydrate structures associated with cell surfaces play a major role in many biologically important mechanisms such as, for example, cell-cell recognition, signal transduction, infection and inflammation mechanisms, and immunological processes.2][3][4] Cells can be physically and biologically distinguished through their surface carbohydrate patterns.This is an important medicinal aspect with regard to specific tumor markers on cell surfaces which often consist of distinct complex oligosaccharide structures. 5Therefore, studying carbohydrate-protein interactions at a molecular level provides a deeper understanding of fundamental biological regulation mechanisms and opens the gate for novel analytical tools or to manipulate such specific processes for therapeutic purposes.Unfortunately, isolation of pure complex oligosaccharides from natural sources in order to study carbohydrate-protein interaction in detail is a rather difficult venture owing to the microheterogeneity of naturally occurring saccharides.Synthetic oligosaccharides, on the other hand, provide for sufficient amounts of pure material for this purpose.However, the chemical synthesis of complex oligosaccharides is still a laborious and often a difficult task, although significant achievements in this field had been accomplished in the past decades.][8][9] For the construction of mimics for complex oligosaccharides, we follow a concept in which simple glycosyl amino acid building blocks are used for the efficient combinatorial preparation of fully glycosylated peptide (glycopeptoid) libraries of the type shown in Figure 1 which, in turn, can bind to carbohydrate-recognizing proteins (Figure .1). [10][11][12][13][14] Figure 1.Glycopeptoids constructed out of monosaccharides A-D bound through spacers (R=H, alkyl, aryl) to a -peptide Asp backbone as oligosaccharide mimics.
In order to study further the influence the -peptide backbone of the aforementioned glycopeptoids may have on the binding characteristics of the respective glycopeptides, we anticipated to prepare a series of bis-glycosylated hexa-peptides with backbones of different flexibility.Here, we describe the chemical synthesis of the -hexapeptide -Ala--Ala-L-Asp--Ala--Ala-L-Asp bearing a D-galactoside and a D-glucoside unit at the Asp moieties.

Results and Discussion
For the preparation of the hexapeptide -Ala--Ala-L-Asp--Ala--Ala-L-Asp we chose a blockwise approach.Therefore we first prepared suitably protected glycosylated tri-peptide blocks -Ala--Ala-(D-Glc or D-Gal)-L-Asp which allowed for simple condensation to give target hexapeptide.Starting from known t-butyl 3-(benzyloxycarbonylamino)propionate 15 (1), catalytic hydrogenolysis afforded crude 2 which was conjugated without further purification with pentafluorophenyl 3-(fluorenylmethoxycarbonylamino)propionate 16 (3) to give dipeptide 4 in 68% yield.Next, the t-butyl ester of 4 was hydrolyzed to give 5 in quantitative yield.The latter dipeptide was sufficiently pure for the next step.Scheme 1. Synthesis of Fmoc-protected dipeptide Fmoc--Ala--Ala 5.
For the preparation of the glycosylated tripeptide block Fmoc--Ala--Ala-(-D-Gal)-Asp-Ot-Bu 7, dipeptide 5 was condensed with the previously described galactosyl building block 6 10 under various conditions (Scheme 2).Table 1 summarizes the conditions and the yields of tripeptide 7. Scheme 2. Conjugation of dipeptides 5 and 8 with galactosyl amino acid derivative 6 to give tripeptide 7 (see also Table 1).
The best yield of tripeptide 7 was obtained with PyBOP, HOBt (Table 1, entry 2).All other condensation reagents gave only low to medium yields.The low yields were due to the formation of several unidentified byproducts during the condensations.Previously, galactosyl amino acid building block 6 could be condensed with pentafluorophenyl-activated amino acid derivatives in high yield. 10Therefore, dipeptide 5 was also converted into the corresponding pentafluorophenyl ester 8 in 95% yield.However, EDCI 17 was used for this step instead of dicyclohexyl carbodiimide (DCC) as described in the original synthesis 16 because higher yields were obtained with EDCI.The activated ester 8 reacted smoothly with 6 to afford galactosylated tripeptide 7 in 67% yield.Here, work up and chromatographic purification was also facilitated because no byproducts which were difficult to separate were formed.Likewise, coupling of dipeptide pentafluorophenyl ester 8 with previously prepared glucosylated asparaginic acid derivative 9 10 gave tripeptide 10 in 57% yield.The latter was further converted into acid 11 by hydrolyzing the t-butyl ester in 10.PyBOP: benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate 18 ; TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate 19 ; HBTU: Scheme 3. Conjugation of dipeptide 8 with glucose building block 9 followed by partial deprotection to give glucosylated tripeptides 10 and 11.
Next, peptide 7 was converted into tripeptide 12 by removing the Fmoc group.Tripeptides 11 and 12 were then coupled under various conditions to give the corresponding galactose-and glucose-containing hexapeptide 13 which finally afforded the free hexapeptide 14 in 71% yield upon complete deprotection (Scheme 4).Table 2 summarizes the conditions for the coupling of 11 and 12 with various standard peptide coupling reagents.
Using EDCI and HOBt as coupling reagent for condensing 11 and 12 did not result in a reaction at all, even under prolonged reaction time (Table 2, entry 1).Raising the temperature above room temperature turned out to be disadvantageous because it only resulted in the formation of unidentified decomposition products which could not be removed.EDCI was also a less efficient coupling reagent for the condensation of 5 and 6 (cf Table 1).Highest yields (61%) of hexapeptide 13 were obtained with the HBTU/HOBt reagent (Table 2, entry 4).PyBOP: benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate 18 ; TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate 19 ; HBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate. 19 b No reaction Complete removal of the protecting groups in hexapeptide 13 was achieved by applying a two step deprotection process.First, the t-butyl ester in 13 was hydrolysed.Next, the Fmoc and actely groups of the crude intermediate were concurrently removed with aqueous ammonia solution to afford the free glucose-and galactose-containing hexapeptide 14 in 71% yield.
For the determination of the secondary structure of 14 in aqueous solution, the CD spectrum and TOCSY and NOESY NMR spectra were measured.The CD spectrum shows a minimum at 202 nm with an ellipticity of -3.5 degree and a maximum at 190 nm with an ellipticity of 8.0 degree.This is indicative for a beta sheet or a random coil secondary structure of hexapeptide 14.1][22][23] Likewise, the chemical shift of the -H of the Asp moieties of 14 at 5.52 ppm indicates a random coil structure for the hexapeptide but is also not significant enough to decide whether 14 adopts a random coil or beta sheet structure. 24,25The TOCSY and NOESY NMR spectra of 14 (Figure 2) show only TOCSY cross peaks but no NOESY cross peaks.Thus, there are only intraresidual long range couplings present but no interresidual ones which proves that 14 adopts a random coil structure.

Conclusions
We have developed an efficient synthetic strategy for the preparation of the bis-glycosylated hexapeptide -Ala--Ala-(-D-Glc)-L-Asp--Ala--Ala-(-D-Gal)-L-Asp 14 and showed this hexapeptide to adopt a random coil conformation in aqueous solution.The synthetic strategy will now be further applied to the chemical synthesis of similar glycosylated hexapeptides containing conformationally more restricted -amino acids instead of -alanine, like for example (1R,2R)-2aminocyclohexane-and cyclopentanecarboxylic acids.Studies toward the conformation and binding of these glycosylated hexapeptides to lectins will be published elsewhere.

Experimental Section
General.All solvents were dried and distilled prior to their use.Reactions were performed under Ar and monitored by TLC on Polygram Sil G/UV silica gel plates from Macherey & Nagel.Detection was affected by charring with H2SO4 (5% in EtOH) or by inspection of the TLC plates under UV light.NMR spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz for proton spectra and 100 MHz for carbon spectra.Tetramethylsilane was used as the internal standard.Signal assignments were confirmed through HH-, CH-COSY, HMBC and HSQC experiments.Conformations were deducted from NOESY and TOCSY experiments and CD spectra, measured on a Jasco J-720 circular dichroism spectrometer using a 1 mm cell.FAB MS was performed on a Finnigan MAT TSQ 70 spectrometer.HRFD MS was performed on a Bruker FT-ICR spectrometer.Elemental analyses were performed on a Hekatech Euro EA 3000 CHN analyzer.Optical rotations were measured with a Perkin-Elmer Polarimeter 341.Preparative chromatography was performed on silica gel (0.032-0.063 mm) from Macherey & Nagel using different mixtures of solvents as eluents.

Scheme 4 .
Scheme 4. Coupling of tripeptides 11 and 12 to afford hexapeptide 13, and full deprotection of the latter to give hexapeptide 14 containing a glucose and a galactose moiety at the Asp residues.

Figure 2 .
Figure 2. Superposition of the TOCSY and NOESY NMR spectra of 14 in 9:1 D2O/H2O; all cross peaks above and below the diagonal belong solely to the TOCSY spectrum; all signals of the NOESY spectrum are located in the diagonal.

Table 1 .
Condensation of dipeptide 5 with galactosyl amino acid derivative 6 under various reaction conditions