The high-mannose C-glycopeptide published b y Wang and co-workers in 1 997 [5] was the first report describing the synthesis of an inhibitor for PNGases. The synthetic scheme employed in the synthesis of this compound used a chemoenzymatic
approach.
In the first part of the synthesis the glucosamine-based building block 5 was synthesised bearing an anomeric ,B-configured aminomethyl-group suitable for the attachment to the side chain of aspartic acid in a pre-assembled peptide.
OH a) Phthalic OAc
�
anhydridelEt3N�
TMSCN HO b) AC20iNaOAc Ac O BF3 · OEt2 00 OH AcO OAc --. NH2 DMF NPhth MeN02 1 HCl 2 3 NPhth b) BOC20 NPhth a) Hydrazine b) AC20, Py 4b c) Hel5
NHAcFigure 3 : Synthesis of a C-glycoside building block for attachment to aspartic acid in pre-assembled peptides reported by Wang et aL [5] .
After the attachment to the side chain of aspartic acid in a pre-assembled
pentapeptide, this molecule was used as an acceptor substrate in the crucial enzymatic transglycosylation reaction to yield the desired high-mannose structure 8 (figure 4). The enzyme used for this reaction step was endoglycosidase A (Endo A), [89-9 1 ]
which hydrolyses an intact glycan from a glycopeptide by cleaving between the two innermost G1cNAc residues of the invariant pentasaccharide core. This enzyme is not yet commercially available and was a gift from Takara Shuzo Co. in Japan to the authors.
Chapter 1 - Introduction 27
In this synthesis, the donor substrate used was a high-mannose glycopeptide (Man9GlcNAc2Asn) that was isolated from soybean flour [92],[93]. During the transglycosylation step, the enzyme cleaves the glycosidic bond between the two innermost GlcNAc residues of the donor. However, instead of simply hydrolysing this glycosidic bond, it also posesses the capability to transfer the reducing end of the carbohydrate chain to the 4-position of the acceptor substrate 7. The reported yield for the resulting high-mannose C-glycopeptide 8 was 26 %. However, the drawback of this synthesis is the extremely limited availability of endoglycosidase A.
8oc-Tyr-IIe-Asn-Ala-Ser-OMe I
Boc-Tyr-l!e-Asp(OH)-Ala-Ser-OMe
�
OAC NH_ AcO 0 /
HBTU HOBt , AcO CH 2 NH<\c 6 Man9GlcNAcrAsn GlcNAcAsn a) NH3,MeOH - b) 3M HCI H-Tyr-lIe-Asn-Ala-Ser-NHz H-Tyr-IIe-Asn-Ala-Ser-N H2
�
I
Ir
NH<\c 7 - Endo-Ar
NH<\c 8Figure 4: Chemoenzymatic synthesis of the high-mannose C-gIycopeptide 8 by Wang et aL [5] .
Such kinetically controlled enzymatic transglycosylations often proceed with low yields and limited regioselectivity. Nevertheless, they can permit a facile access to the desired product within one reaction step and without the extensive use of protecting group chemistry. Therefore, even if low yielding, they offer a considerable advantage over conventional chemical methods. The concept of enzymatic transglycosylation is well known [94] .
Before conducting inhibition experiments using the high-mannose C-glycopeptide, the compound was tested as a substrate for the PNGases to be used in the inhibition trials (see below). The authors reported that none of the PNGases used was able to hydrolyse the C-glycopeptide.
Chapter 1 - Introduction
This synthetic high-mannose C-glycopeptide was shown to have Kj- values of 1 and 43 llM for PNGase A and F respectively. The substrate used for these experiments was the corresponding N-glycopeptide. The Ki-values with PNGase Srn
(Sphingobacterium multivorum) and PNGase HO (hen ovalbumin) were determined using a 14C-labelled fetuin glycopeptide and calculated to be 1 52 and 1 57 llM respectively.
28
The authors concluded that their synthetic C-glycopeptide was an inhibitor of glycoamidases from various sources and could therefore be termed a broad-spectrum inhibitor for this class of enzymes.
A year later in 1 998 a different research team, but again supervised by Yuan C. Lee [6], reported the synthesis of a high-mannose glycopeptide analo gue containing a glucose-asparagine linkage (figure 5). Again the approach consisted of the
straightforward synthesis of a peptide linked acceptor substrate which was then used in an enzymatic transglycosylation reaction employing endoglycosidase A. This approach was completely reminiscent of the synthesis of the high-mannose C
glycopeptide with the only difference being that the acceptor substrate used in the enzymatic step was a glucose N-linked to a pentapeptide.
G1cNAcAsn H-Tyr-Ile-Asn-AJa-Ser-NH2
��o
Endo-A H-Tyr-Ile-Asn-AJa-Ser-NHJ OH OH 9 10Figure 5: Enzymatic transfer of the donor Man9GlcNAc to the glucosyl pentapeptide acceptor using endoglycosidase A by Deras et aL [6].
As with the high-mannose C-glycopeptide this glycopeptide mimetic was tested as a substrate for commercially available PNGase A and F. Neither of the enzymes were capable of hydrolysing this compound.
Chapter 1 - Introduction 29
Inhibition experiments were performed with 10 (figure 5) testing its inhibitory activity in a digest of a glycopeptide isolated from bovine asialofetuin. The Ki values were determined to be 2 �M for PNGase A and 53 �M for PNGase F. The authors do not state why they tested the high-mannose glycopeptide mimetic against a substrate bearing complex glycans rather than the high-mannose glycan that had been used in the experiments with the C-glycopeptide.
The authors draw the conclusion that the acetamido group of the innermost sugar plays an important role in the recognition and cleavage of N-glycosidic bonds by PNGases. Furthermore, the inhibition constants are similar to those calculated for the inhibition of PNGase A and F using the C-glycopeptide. However, they do not take into consideration the fact that different substrates had been used in both inhibition trials.
A very interesting result reported by the authors is that the truncated glycopeptides Man9GlcNAc2Asn and Man9GlcNAcGlcAsn as well as the free glycan from fetuin glycopeptide weakly inhibited PNGase F
(Ki
� 1 mM) but not PNGase A. Conversely, a shortened version of the glycopeptide mimetic having only a single glucose on the peptide backbone did not show any inhibitory effect. Thus the authors hypothesise an important role for the oligosaccharide chain in the inhibition of PNGases.Haneda and Tanabe from the Noguchi Research Institute in Japan described the synthesis of a complex glycopeptide derivative N-linked to the side chain of
glutamine and the testing of its inhibitory effect towards PNGase F in their Japanese patent of November 2000 [7] .
As was the case for the two inhibitors previously described, this synthesis was also achieved using a chemoenzymatic approach employing an endoglycosidase. This time the authors prepared a glycopeptide as an acceptor substrate which had a single GlcNAc bound to the side chain of glutamine in the hexapeptide (H-Lys-Val-Ala Gln(GlcNAc)-Lys-Thr-OH). Transglycosylation using endoglycosidase M from Mucor hiemalis [95],[96] and a disialo complex-type donor glycopeptide produced the glutamine linked glycopeptide mimetic 1 2 shown in figure 6.
Chapter 1 - Introduction 30
(NeuAcGaIGlcNAcMan)zManGlcNAcz-Asn GIcNAc-Asn
B·L" .v
Endo-M
1 1
Figure 6: Synthesis of the glutamine bound disialo glycopeptide 1 2 using endoglycosidase M by Haneda and Tanabe [7].
Nmc
1 2
This newly synthesised glycopeptide mimetic was tested as a substrate for both PNGases A and F . The authors reported that neither of the enzymes were capable of hydrolysing this glycopeptide.
Inhibition experiments conducted using the corresponding Asn-linked glycopeptide as a substrate showed that in a 1 : 1 mixture of the Asn and the GIn-linked glycopeptide the activity of PNGase F was reduced to 77 % compared to the control without any
GIn-bound inhibitor present. No inhibition constants have been reported for this
compound towards PNGase F nor has any kinetic data been reported with PNGase A.
The authors conclude that their GIn-linked glycomimetic is stable to the in vivo degradation by PNGases and therefore offers some potential in the design of physiologically stable glycopeptide based drugs. The potential for this class of mimetics to act as inhibitors of PNGases (PNGase F) may therefore be valuable in the future.