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Manual and automated syntheses of the N-linked glycoprotein core glycans Salvatore G. Pistorio, Scott A. Geringer, Keith John Stine, and Alexei V. Demchenko J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b03056 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 11, 2019

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The Journal of Organic Chemistry

Manual and automated syntheses of the N-linked glycoprotein core glycans

Salvatore G. Pistorio, Scott A. Geringer, Keith J. Stine, and Alexei V. Demchenko*

Department of Chemistry and Biochemistry, University of Missouri – St. Louis One University Boulevard, St. Louis, Missouri 63121, USA e-mail: [email protected]

Abstract: Presented herein are two complementary approaches to the synthesis of the core Nglycan pentasaccharide. The first, a traditional manual approach in solution, makes use of the H-bond-mediated aglycone delivery (HAD) method for highly diastereoselective introduction of the β-mannosidic linkage at room temperature. The synthesis of the core pentasaccharide was also accomplished using an HPLC-assisted automated approach. The overall assembly was swift (8 h) and efficient (31%).

Keywords: glycosylation, synthesis, oligosaccharides, automation, solid phase

1 ACS Paragon Plus Environment

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Introduction Glycans and glycosylated proteins (glycoproteins) play important roles in various biological processes and the general interest in these compounds and their functions has rapidly increased in the recent years.1-6 Among a plethora of structures, N-glycans and other oligosaccharides are covalently attached to proteins at asparagine (Asn) residues by an N-glycosidic bond. NGlycans are also found on the surface of a variety of pathogens and are involved in mediation of the pathogenesis of cancers,7 AIDS,8 Alzheimer’s disease,9 and other processes.10,11 All Nglycans are classified into three general classes: high-mannose, hybrid, and complex,2 but all share Manα16(Manα13)Manβ14GlcNAcβ14GlcNAcβ1Asn, the common core sequence (Figure 1). Figure 1. The core pentasaccharide sequence of N-glycans HO HO HO

OH O O HO O

HO HO HO

OH O

NHAc O HO O OH

HO O

OH O

O OH

H N NHAc N H

O

O

The construction of the core structure as the key intermediate towards a diverse library of N-glycans has been a vibrant area of research. Chemical and chemoenzymatic syntheses of the N-glycan core pentasaccharide have been reported by Ogawa,12 Unverzagt,13-15 Ito,16-18 Schmidt,19,20 Seeberger,21,22 Danishefsky,7,8,23,24 Boons,5,25 L.-X. Wang,26 Fukase,27,28 Wong,29 P. G. Wang,30 amongst others. Nevertheless, the construction of these glycan sequences remains a notable challenge, which slows biomedical studies related to understanding the roles of glycoproteins and creation of N-glycan-derived pharmaceuticals. Described herein is two distinct approaches to the synthesis of the N-linked core pentasaccharide 1 (Scheme 1). One approach was based on the traditional linear sequential synthesis in solution, whereas the 2 ACS Paragon Plus Environment

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second approach was based on the convergent assembly using the HPLC-based oligosaccharide synthesizer developed in our labs.

Results and Discussion One of the challenges of the chemical synthesis of N-glycans is the introduction of the Manβ(14)GlcNAc unit present at the branching point of the core sequence.31,32 A strong anomeric effect in mannosides, and the impossibility of using the neighboring group participation to aid in the synthesis of the β-linkage in the manno series, make this bond one of the most difficult steps in the synthesis.32 Some promising methods have been established by Crich33-39 and others,40-42 and the approach developed by us also allows achievement of excellent stereocontrol in β-mannosylation.43 Our approach relies on the use of picoloyl (Pico) protecting groups at remote positions capable of controlling the stereoselectivity via the Hbond-mediated Aglycone Delivery (HAD).44-46 According to this approach, the hydroxyl of the glycosyl acceptor forms the hydrogen bond with the picoloyl nitrogen of the glycosyl donor. As a result, the nucleophile is delivered from the same (syn) face of the sugar ring in respect to the picoloyl group. For compounds of the D-manno series, excellent stereocontrol could be achieved with picoloyl groups at C-3 and/or C-6 positions and a series of β-mannosides have been obtained with high stereoselectivity and yields.43 Hence, we decided to investigate whether this approach would also be suitable for the synthesis of the core pentasaccharide sequence of N-glycans. For this synthesis, three building blocks (2-4) were selected in accordance of the retrosynthetic analysis of the pentasaccharide target 1 depicted in Scheme 1. Among these, building block 447 was chosen for introducing the two glucosamine units at the reducing end of the sequence. Building block 343 equipped with two picoloyl protecting groups at positions C-3 and C-6 was selected to carry out the HAD-assisted synthesis of the Manβ(14)GlcNAc



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linkage. Since the picoloyl groups can be selectively removed in the presence of many other protecting groups used in carbohydrate chemistry, this 3,6-di-O-Pico substitution would offer a straightforward access to subsequent α-mannosylation. For the purpose of introducing the two terminal α-mannosyl residues, building block 248 was selected. Finally, we chose to equip our target molecule with 4-azidobutyl spacer to offer a convenient access to selective conjugation or surface immobilization of the final product. Scheme 1. Retrosynthetic analysis for synthesis of the target pentasaccharide 1 from building blocks 2-4. HO BzO BzO BzO 2

OBz O

HO HO

OH O O HO

SEt HO HO HO

OH O

AcHN

HO

O

O HO

HO O

O

O O AcHN

HO

NH2

1

O OH PicoO BnO PicoO 3

Ph

OBn O SEt

O O BnO 4

O SEt NPhth

In accordance with the retrosynthetic analysis, the assembly of the target pentasaccharide 1 started with the universal precursor 4 that was used to introduce both glucosamine units. First, compound 447 was converted into glycosyl donor 5 that was reacted with 4-azidobutanol49,50 to give the functionalized monosaccharide 6 in 82% yield (Scheme 2). Second, glycosylation of acceptor 6 with donor 4 led to the formation of disaccharide 7 in 87% yield. The subsequent reductive opening of the benzylidene acetal afforded compound 8 in 94% yield. Disaccharide 8 was then used as the glycosyl acceptor for β-mannosylation.


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Scheme 2. Synthesis of disaccharide acceptor 8 OBn O

HO BnO

SEt NPhth

HO

N3 NaCNBH3, HCl-Et2O NIS/TfOH, 5 THF, MS 4Å 1,2-DCE, MS 4Å 1 h, 88% 10 min, 82% O Ph O OBn O O SEt BnO HO O NPhth BnO N3 4 NPhth NIS/TfOH, 6 1,2-DCE, MS 4Å 5 min, 87% BnO O Ph O

OBn O

PhthN O

O BnO

O NPhth

7

N3

NaCNBH3, HCl-Et2O 1 h, 94% THF, MS 4Å BnO HO

OBn O

PhthN O O BnO OBn 8

O NPhth

N3

The next synthetic step, the introduction of the β-linked mannose unit, represents the key step in the entire synthesis of the N-glycan pentasaccharide. Somewhat unexpectedly, all attempts to glycosidate 3,6-di-O-Pico ethylthio glycosyl donor 3 with acceptor 8 by varying the amount of the donor as well as type and amount of the promoter have been unsuccessful (entries 1-5, Table 1). None of the attempts led to the formation of the desired trisaccharide intermediate 9. Hence, next we decided to utilize another known donor, 3-O-Pico ethylthio glycoside 1043 for the introduction of the β-linked mannose unit. Initially, our attempts were unsuccessful, and conventional reaction conditions for the HAD reaction43 did not work (entries 6 and 7). Success was achieved when we decided “to push” the reaction and added a large excess of N-iodosuccinimide (NIS, 6 equiv in respect to the donor). Under these reaction conditions, the coupling of glycosyl donor 10 and acceptor 8 produced trisaccharide 11 in 50% yield with excellent stereoselectivity (α/β = 1/11.2, entry 8). Encouraged by this progress, we continued optimizing the reaction conditions and discovered that using 3 equiv of donor 10 along with 3 equiv. of NIS (in respect to the donor) seemed necessary to bring the reaction to 5 ACS Paragon Plus Environment


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completion. As a result, trisaccharide 11 was obtained in a high yield of 92% and commendable stereoselectivity (α/β = 1/12, entry 10). It should be noted that this reaction was performed at ambient temperature, which offers an important experimental advantage over other known methods for β-mannosylation that require very low temperatures.32,38,51-53 Table 1. Optimization of the β-mannosylation of disaccharide acceptor 8 R 6O R 4O PicoO

OBn O

glycosyl donor

3: R4 = Bn, R6 = Pico 10: R4,R6 = >CHPh R 6O R 4O PicoO

glycosyl acceptor 8

SEt

promoter CH2Cl2

OBn PhthN O BnO O O O BnO OBn

OBn O O NPhth

N3

9: R4 = Bn, R6 = Pico 11: R4,R6 = >CHPh

Entry

Donor (equiv to acceptor)

Promoter (equiv to donor)

Product (yield, α/β ratio)

1

3 (1.1)

NIS (2.0), TfOH (0.2)

9, no reaction

2

3 (1.3)

NIS (2.0), TfOH (0.2)

9, no reaction

3

3 (3.0)

NIS (3.0), TfOH (0.3)

9, no reaction

4

3 (1.3)

DMTST (2.0)

9, no reaction

5

3 (1.3)

MeOTf (4.5)

9, no reaction

6

10 (1.1)

NIS (2.0), TfOH (0.2)

11, no reaction

7

10 (1.1)

DMTST (2.0)

11, no reaction

8

10 (1.1)

NIS (6.0), TfOH (0.3)

11 (50%, α/β = 1/11.2)

9

10 (1.3)

NIS (6.0), TfOH (0.3)

11 (56%, α/β = 1/10.0)

10

10 (1.6)

NIS (4.0), TfOH (0.3)

11 (61%, α/β = 1/13.1)

11

10 (3.0)

NIS (3.0), TfOH (0.3)

11 (92%, α/β = 1/12.0)

With trisaccharide 11 in hand, we continued the synthesis of the target pentasaccharide core, and our altered synthetic plan is depicted in Scheme 3. Thus, 3”-O-picoloyl group in 11 was removed with NaOMe in MeOH-CH2Cl2. The resulting trisaccharide acceptor 12 was 6 ACS Paragon Plus Environment

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glycosylated with mannosyl donor 2 in the presence of NIS/TfOH to afford tetrasaccharide 13 in 91% yield. The latter was subjected to acid hydrolysis of the 4”,6”-O-benzylidene acetal to afford tetrasaccharide acceptor 14 in 88% yield. The latter was regioselectively glycosylated with mannosyl donor 2 to afford the protected N-linked core pentasaccharide 15 in 60% yield. Scheme 3. The final assembly of the N-glycan pentasaccharide core 15 Ph BzO BzO BzO 2

OBz O

O O RO

SEt

NIS/TfOH, 1,2-DCE,MS 4Å RO


RO O BzO BzO BzO

OBn OBn PhthN O BnO O O O O O BnO NPhth OBn 11: R = Pico 12: R = H NaOMe/ MeOH DCM 1h, 86% 20 min, 91%

O OBz

13: R = >CHPh 14: R = H

2 BzO BzO BzO

OBz O

NIS/TfOH, 1 h, 60% 1,2-DCE,MS 4Å

O


HO O BzO BzO BzO

O OBz

N3

OBn O O NPhth

N3

TFA/DCM 88%

OBn O O NPhth

N3

15

Having achieved the linear synthesis of the core pentasaccharide in solution, we decided to investigate whether the HPLC-assisted oligosaccharide synthesis, an automated approach developed in our labs,54,55 would also be a suitable mode for obtaining this challenging branched structure. The traditional solution phase synthesis is an important method to obtain oligosaccharides, but also the solid-phase automated approach has become a viable means to obtain glycan sequences.56 Following the pioneering work by Seeberger and co-workers who have been the first to develop an automated solid-phase oligosaccharide synthesizer,57-62 several other research groups63-72 have been developing other platforms. These efforts have already demonstrated that the automation is a viable alternative for obtaining complex



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glycans.56 Hence, the synthesis of the N-linked core glycan appealed to us as an interesting challenge, particularly since our method has not yet been applied to the synthesis of branched glycan sequences. Scheme 4. Preliminary attempt to obtain Manβ(1-4)GlcNAc-linked disaccharide 21 on solid phase using the HPLC synthesizer BnO HO BnO

O HO

SEt PhthN

4

5

OBz

Bn N

O O

16

NIS/TfOH 92% PhthN BnO HO

O

3

O

BnO

17

O O

BnO

O O

1) NaOMe/MeOH 2) Succinic anhydride, Py 78% 2 steps O

Bn N

PhthN BnO HO

OBz

Bn N

3

O

O

OH

O

O

18

H 2N EDC, DMAP

BnO HO

NPhth O

O

3

O

Bn N

O O

BnO

O

PicoO

20 TMSOTf/DCM

Washing DCM

BnO O BnO

OPO(OBu)2

Cleavage: NaOMe/MeOH/DCM

Washing DCM, MeOH, DCM OBn O

0.11 mmol/g

OBn O

BnO PicoO

AcO AcO

H N

19

Auromated Assembly

AcO

O

Ac2O/Py

NPhth O

O

3

Bn N

OAc O O

 = 1/1 86%

21

Since the pentasaccharide target contains a challenging Manβ(1→4)GlcNAc linkage, we initially performed a number of model β-mannosylations on solid phase. For this purpose, we obtained the resin-bound glycosyl acceptor 19 as depicted in Scheme 4. Known 8 ACS Paragon Plus Environment

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glucosamine derivative 547 was coupled with spacer 16 in the presence of NIS/TfOH to afford glucosamine-spacer intermediate 17 in 92% yield. Benzoyl deprotection followed by the treatment with succinic anhydride in pyridine allowed us to obtain building block 18. The latter was coupled with the amine of JandaJel resin in the presence of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) to afford building block 19. The loading capacity of JandaJel functionalized acceptor 19 was determined to be 0.11 mmol/g by cleaving off and quantifying the loaded compound. Although we managed to obtain very high yields in glycosylation of 19 with phosphate donor 20, ultimately, these attempts were deemed unsuccessful due to poor stereoselectivity observed in these reactions. As a result, disaccharide 21 was obtained in 86% yield albeit no stereoselectivity. Since the direct stereoselective formation of the β-mannosidic linkage on solid phase has failed, we explored an alternative approach wherein the Manβ(1-4)GlcNAc linkage was presynthesized in solution and the resulting disaccharide building block was used for the indirect introduction of the β-mannosidic unit on solid support. Thus, our subsequent plan to assemble the N-glycan core pentasaccharide 22 using the HPLC-assisted automated approach involved building blocks 19, 23 and 24 depicted in Scheme 5. Scheme 5. Retrosynthetic analysis of the solid-supported pentasaccharide target 22 BzO BzO BzO BzO

OBz O

BzO BzO

O BnO O

OPO(OBu)2 23

1 OBz O

BzO BzO BzO PicoO BnO PicoO 24

OBn O BnO O BnO

NPhth O

O BnO

22

O OBz

OBn O BnO O

NPhth OTCAI O

HO BnO

OBn O

O NPhth

OBn O

O NPhth

BnO


Bn N 4

O

O

O

Bn N 4 O 19

H N

O O

O O

H N

O O


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As shown in Scheme 6, the synthesis of disaccharide building block 24 began by glycosylation of acceptor 2573 with donor 10. To ensure the completeness of the reaction we had to use excess of donor and promoter as shown in Scheme 6. Under these conditions, we were able to obtain disaccharide 26 in excellent yield of 95% and commendable β-manno stereoselectivity (α/β = 1/6.0). Picoloyl deprotection was affected using a 1 M solution of NaOMe in MeOH to afford compound 27. This step was necessary to ensure the regioselectivity of the subsequent reductive opening of the benzylidene acetal that otherwise was non-regioselective. As a result, disaccharide 28 was obtained with high regioselectivity and 77% isolated yield. The later was 3,6-di-picoloylated to give 29 in 95% yield. Compound 29 was then treated with TBAF to afford hemiacetal 30 in 60%. Finally, compound 30 was converted into trichloroacetimidate donor 24 in 83% yield by reaction with trichloroacetonitrile in the presence of DBU. Scheme 6. Synthesis of disaccharide donor 24 for the automated assembly Ph

OBn O

O O PicoO 10 2.0 equiv

BnO HO BnO

SEt

95%   1/6.0

Ph

OBn O BnO O

O O PicoO 26 HO

28 PicoO

OBn O BnO O

BnO PicoO 30

OTBS NPhth

25

NIS/TfOH (4.0/0.40 equiv) CH2Cl2 NPhth O

OTBS

NaOMe/MeOH Ph 98%

BnO

OBn O BnO O BnO

BnO HO

O

OBn O BnO O

O O HO

BnO

27 NPhth OTBS O

NPhth OH O

PicoOH, PicoO OBn DMAP, EDC O BnO BnO PicoO O 95% BnO 29 PicoO CCl3CN, DBU 83%

BnO PicoO

BnO

24

NPhth OTBS O

OBn O BnO O

Cu(OTf)2 BH3 THF 77%

NPhth TBAF/THF OTBS 60% O

NPhth OTCAI O

BnO

The automated assembly shown in Scheme 7 followed typical cycles of glycosylation and deprotection steps with the interim washing off the excess reagent, similarly to that used 10 ACS Paragon Plus Environment

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in our previously described automated oligosaccharide synthesis.55 JandaJel polymeric resin functionalized with glycosyl acceptor 19 was packed in OmnifitTM glass chromatography column. The column was integrated into the standard Agilent Infinity 1260 HPLC system equipped with a quad-pump and an autosampler, and the automated assembly was programmed as follows. To affect the initial washing and swelling of the resin containing acceptor 19, Pump D was programmed to deliver dichloromethane. After that, Pump C was programmed to deliver donor 18 in CH2Cl2, and the resulting solution was recirculated for 10 min. The delivery of the donor has been monitored with the UV detector that showed a plateau at the end of the recirculation. The autosampler was programmed to deliver a solution of TMSOTf in CH2Cl2 (3 injections) and the resulting reaction mixture was recirculated for 90 min. The glycosylation has been monitored with the UV detector that showed a steady change in absorbance of the reaction mixture due to the consumption of glycosyl donor 18. When the UV-monitoring showed no further change in absorbance of the eluate passing through the detector, the system was washed with CH2Cl2 (Pump D). Subsequent deprotection of the two picoloyl groups from positions C-3” and C-6” was then performed by delivering the deprotection reagent copper(II) acetate in methanol/dichloromethane using Pump A for 20 min. The deprotection was monitored with the UV detector that showed a steady change in absorbance of the reaction mixture due to the release of the Pico protecting groups. When the UV-monitoring showed no further change in absorbance of the eluate passing through the detector, the system was washed with CH2Cl2 (Pump D for 10 min). The final branching via di-mannosylation was conducted by delivering mannosyl donor 17 (Pump C). This monitored reaction was repeated two times with the interim washing (Pump D) to ensure the complete glycosylation of both hydroxyls of the acceptor. At the end of this automated sequence, the immobilized pentasaccharide 16 was cleaved from the polymer support by delivering a mixture containing a 1 M solution of NaOMe/MeOH in CH2Cl2 and MeOH with Pump B for 10 min. The eluate was collected,



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neutralized, concentrated, and the residue was acetylated with Ac2O in pyridine to afford pentasaccharide 20 in 31% yield overall. Scheme 7. Automated assembly of pentasaccharide 22 and its cleavage from the solid support to produce compound 31 HO BnO

OBn O O NPhth Swelling DCM

Bn N

O O

4

H N

O O

O

19 24, TMSOTf/DCM

Washing DCM Cu(OAc)2/MeOH/DCM

Washing DCM

23, TMSOTf/DCM

Washing DCM Washing DCM

23, TMSOTf/DCM

22 NaOMe/MeOH/DCM

AcO AcO AcO

OAc O O BnO O

AcO AcO AcO

O OAc

Ac2O/Py OBn O

BnO O

NPhth O BnO O OBn 31

OBn O O NPhth

4

Bn N

O

OAc

O

With similarly protected pentasaccharides 15 and 20 in hand, we performed the global deprotection of compound 15 as shown in Scheme 8. O-Acyl protecting groups were removed by treatment with NaOMe/MeOH. The resulting crude product was then subjected to the treatment with 1,2-ethylendiamine (EDA) in n-butanol-toluene to affect the removal of the Nphthaloyl groups. The crude deprotected product was then per-acetylated with acetic anhydride in the presence of pyridine to afford compound 21 in 90% yield over three steps. The latter was subjected to the Birch conditions (Na/liq. NH3) that affected the global deprotection by removing benzyls, acetates, and reducing the azide group into amine. The fully deprotected Nglycan pentasaccharide core 1 was isolated in 66% yield. It should be noted that the


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deprotection of 20 can be performed similarly and will require essentially the same sequence of synthetic steps. Scheme 8. Deprotection of compound 15 to afford the target N-glycan 1 15 AcO AcO AcO

OAc O O AcO O

AcO AcO AcO

O OAc

1) NaOMe, MeOH 90% 2) EDA, n-Butanol-toluene 3) Ac2O, Py, DMAP OBn AcHN O BnO O O O BnO OBn 32

OBn O O NHAc

N3

Na, liq. NH3, THF

66% 1

Conclusions N-Linked glycan core pentasaccharide was assembled using the solution-based manual and solid-phase automated approaches. Both syntheses produced the desired core structure with high efficiency and comparable outcome for the assembly stage. This was our first attempt to obtain the branched oligosaccharide using the automated approach and the overall assembly was swift (8 h). High diastereoselectivity for β-mannosylation was achieved at room temperature using the H-bond-mediated aglycone delivery (HAD) method in solution. The HAD method on solid phase yielded no stereoselectivity for -mannosylation, which has been by-passed by using the convergent approach. According to this approach, we obtained the disaccharide building block containing challenging Manβ(1→4)GlcNAc bond that was then integrated in our HPLC-based automated oligosaccharide assembly.



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Experimental General Methods. Column chromatography was performed on silica gel 60 (70-230 mesh), reactions were monitored by TLC on Kieselgel 60 F254. The compounds were detected by examination under UV light and by charring with 10% sulfuric acid in methanol. Solvents were removed under reduced pressure at

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